Fellows Research Projects

Project ESR1

Project Title: From endocytosis to transcytosis: investigation of key steps and signals
Fellow’s Name: Diana Hudecz
Tutor’s name: Prof. Kenneth A. Dawson (NUID UCD); Co-supervisor: Marino Zerial (MPI-CBG)

Project Description:
The main goal of this ESR project is to understand the fundamentals behind the uptake mechanism and intracellular trafficking of nanoparticles within and across the blood-brain barrier (BBB). The project will utilize novel dual targeting strategies with protection of the targeting moieties from non-specific protein binding, whereby one targeting moiety will be used to recognize and adhere to the blood-brain barrier, and a second will be utilized to drive the nanoparticle out of the endolysosomal pathway and towards the transcytotic pathway (e.g. ApoE or transferrin). Specific objectives are to develop a standardized platform for determining endocytosis/transcytosis efficacy for nanomaterials, and to identify targeting strategies that works in vitro.

As part of this Marie Curie, program an emphasis will be placed upon collaborative aspects with other ESR/ERs across the project as the work builds. Research secondments at the partner and other institutes (MPI-CBG, ACS) will form part of the training experience while enhancing the overall output quality.

Methodology/Skills associated with project:
Set up and optimisation of different in vitro BBB models; different imaging techniques, such as spinning disc-, confocal-, fluorescence-, and electron microscopy, as well as other methods, to quantify nanoparticle localization and passage through barriers. Significant focus will be placed on gaining quantitative reproducible experimental data to disentangle the detailed mechanism by which the nanoparticles are transported.
Project ESR2

Project Title: Harnessing Natural Pattern Recognition Mechanisms for Macromolecular Drug Delivery
Fellow’s Name: Martina Tuttolomondo
Tutor’s name: Prof. Dr. Jan Mollenhauer, Prof. Kenneth A. Dawson

Project Description:
Pattern recognition is a fundamentally simple and evolutionary ancient mechanism, by which host proteins can recognize an extremely broad range of ligands and elicit corresponding responses of mainly protective nature. Until recently, pattern recognition has been regarded as underlying the defense against a broad range of bacterial and viral pathogens only. Our work, however, demonstrated that the same principles also guide regenerative processes by modulation of signaling in the extracellular matrix. The functional diversity of single human pattern recognition molecules such as DMBT1 may at least in part depend on the recognition of fundamentally simple structures, including poly-phosphated and –sulfated ligands. Interaction of the pattern recognition molecule DMBT1 or of short peptide mimics with ligands results in self-assembling complexes, which is probably based on pi-stacking similar to the amyloid fibril formation in Alzheimer`s disease. But in this case the effect is anti-pathogenic not pro-pathogenic. Moreover, ongoing work suggests that adsorption of pattern recognition molecules can under certain circumstances attach a stealth character and aid to circumvent eliciting immune responses, while simultaneously promoting uptake by epithelial cells. The major goal of this project is to use pattern recognition mechanisms and modules for the rational design of macromolecular drug delivery devices, which are biocompatible, biodegradable and non-immunogenic.

Methodology/Skills associated with project:
The ability of DMBT1/peptide mimics to self-assemble macromolecular drugs (e.g. nucleic acid-based drugs), their cell type selectivity and ability to deliver macromolecules to and across epithelial cells is analyzed. Uptake by and cargo delivery to epithelial cancer cells, specifically cancer stem cells, will be analyzed in in vitro model systems.
Project ESR3

Project Title: Interaction of nanoparticles with the pulmonary barrier; the role of mucus in health and disease
Fellow’s Name: Xabier Murgia Esteve
Tutor’s name: Prof. Dr. Claus-Michael Lehr, Dr. Cristiane de Souza Carvalho

Project Description:
This project aims to develop a new cell culture model of the upper respiratory tract, minimally comprising epithelial cells and mucus, and possibly other elements such as mucus producing cells, macrophages, dendritic cells, and others, that allows us to study the deposit aerosolized nanoparticles (e.g. to be developed by the project partners) at an air-interface, that is the more close condition for in vivo situation. With this model we aim to study the role of mucus and surfactant as non-cellular elements of the pulmonary barrier, addressing pathological changes in such model and evaluating the nanoparticle transport under the influence of some diseases such as inflammation and/or bacterial infection. Overall this model will contribute to better understand the physiological and pathological factors responsible for nanoparticles transport through bronchial mucus and eventually across the air-blood barrier.

Methodology/Skills associated with project:
Set up a cell culture model of the airway epithelium that develops mucus gel layer and analyze it under different physiological conditions, including the interaction between mucus-nanoparticles, comparing binding and possible uptake by epithelial cells and macrophages, and thus address the role of mucus on the physiological and pathological conditions.
Project ESR4

Project Title: Apolipoprotein E and A targeted albumin nanoparticle delivery to the CNS: Mechanism and route of delivery
Fellow’s Name: Anne Iltzsche
Tutor’s name: Dr. David Begley (KCL)

Project Description:
This project aims to elucidate the entry mechanism and routing of apolipoprotein-targeted albumin nanoparticles to neurons of the central nervous system across the blood-brain barrier. Questions that will be addressed are whether the subsequent nanoparticle transport within the brain occurs via the intracellular route. This will be studied using carefully timed electron microscopy after intravenous nanoparticle administration. Furthermore, we will investigate whether the albumin nanoparticles present in the cytoplasm of neurons still have their apolipoprotein target attached or if it was removed during transcytosis. We aim to use the albumin nanoparticles for the delivery of radiolabelled drugs and furthermore compare different nanoparticle systems of NUID UCD, UNIMA and EPOS head to head regarding their drug delivery ability.

Methodology/Skills associated with project:
A combination of in vivo and in vitro techniques developed at KCL will be employed to study nanoparticle transport across the blood-brain barrier. Comparison of different transport vectors in use at NUID UCD, UNIMA and EPOS aiming to investigate their ability to deliver radiolabelled drugs to the CNS.
Project ESR5

Project Title: Optimizing the transport of nanoparticles through mucus secreting cancers.
Fellow’s Name: Ahmed Eladly
Tutor’s name: Dr. Andreani Odysseos

Project Description:
Mucus associated with cancer acts as a tenacious barrier that attenuates the diffusion of most therapeutic and diagnostic moieties leading to suboptimal therapeutic outcome. Mucin is a glycoprotein-based structure made up of a protein backbone with carbohydrate chains attached at intervals arranged into a bottle brush configuration. Mucinous adenocarcinomas are malignancies that originate from glandular epithelium of different host tissues including the lung, breast, colon, prostate, or pancreas and are approximately more than 50% mucus in terms of total tumor mass. In most cancers, there is over-secretion of mucus giving rise to thicker layers and heavily intertwined carbohydrate chains. Brush-like mucin extensions trap and hinder macromolecular drugs and nanoparticles (NP) and obstruct drug-cell interaction. Therefore we aim to mechanically probe at the nano-scale the mucus barriers in cancer using a novel atomic force microscope technique. This will give us clues into the mechanical properties of mucus barriers to help design optimal drug delivery systems and enable a mechanistic comparison between mucus in cancer and in healthy state.
Also, we will develop and validate a mathematical model that can predict the diffusion of NPs of defined physicochemical properties across cancerous mucosal barriers based on steric, hydrodynamic, electrostatic and mechanical data obtained from in vitro experiments. Lastly, the model will be used to identify design criteria for NPs that would afford optimal penetration through mucus in malignancies.

Methodology/Skills associated with project:
1. Establishment of mucus secreting in vitro cell culture and three dimensional tissues constructs.
2. Utilization of atomic force microscopy to study mechanics of mucus and cells (sample preparation, calibration, force measurements model fitting)
3. Application of multiple particle tracking/fluorescent microscopy and other in-house staining techniques to obtain information about the diffusion trajectories of NP through cancer mucus.
4. Implementation of finite element modeling to describe the microstructure of mucus using representative volume elements (RVE).
5. Adoption of Stokesian dynamics method and a random walk approach to simulate electrostatic interactions and diffusion of NP respectively.
Project ESR6

Project Title: Development of bio-inspired routes for delivery of siRNAs
Fellow’s Name (if hired):
Tutor’s name: Marino Zerial, Colin Wilde

Project Description:
The objective of this project is to address the key bottlenecks in the delivery of macromolecules (focusing on siRNA delivery) to develop innovative therapeutical systems using a bio-inspired approach. In the case of siRNA, delivery is a multi-step process including: (1) stabilization and bioavailability of the formulation, (2) targeting to specific tissues and cells, (3) internalization from the plasma membrane, (4) intracellular trafficking routes and (5) release/escape from intracellular (mostly endosomal) compartments into the cytosol, (6) loading onto an RNA-induced silencing complex (RISC). Despite the fact that in the last years several successful approaches have been developed to improve some of the above steps, siRNA delivery remains an inefficient process that has not yet resulted in clinical applications. The Zerial group has accumulated over two decades a profound understanding of the molecular mechanisms underlying endocytosis, endosome biogenesis, transport and maturation. We recently used lipid nanoparticles (LNPs) loaded with traceable siRNAs to monitor delivery to different cell types in vitro and in mouse liver by quantitative fluorescence and electron microscopy. We estimated that the escape of siRNAs from endosomes into cytosol is only 1-2% and occurs only within a limited time window corresponding to a specific compartment sharing early and late endosomal characteristics (Gilleron, Zerial et al., Nature Biotechnology 2013). We have also synthesized artificial endosomal vesicles recapitulating the activity of membrane fusion displayed by the cellular organelles. In this project, we will use our established pipeline to functionally characterize novel formulations and provide a proof of principle for the exploitation of endocytic trafficking mechanisms to improve siRNA delivery for therapy.

Methodology/Skills associated with project:
1. Visualize the intracellular fate of siRNA formulations in vitro and in vivo;
2. Monitor siRNA escape from endosomes;
3. Screen chemical compounds and RNAi libraries in high content cell-based image analysis assays;
4. Apply image analysis and statistics to validate the candidate formulations.
Project ESR7

Project Title: Nanoparticle exposure of placental barriers – an indirect route of teratogenesis?
Fellow’s Name: Catherine Gilmore
Tutor’s name: Dr Patrick Case

Project Description:
Previous research within our laboratory has used an in vitro model of the placental barrier (between mother and fetus) to investigate whether there might be any theoretical risk of toxicity of nanoparticles (NPs) during pregnancy. The exposure of this model barrier in tissue culture showed that there was indirect damage to cells beneath the barrier. This was not mediated by NPs passing through the barrier but by a process of signalling both within and from the barrier after NP exposure. In this project, in order to further investigate the correlation of these results with the in vivo situation in humans, we aim to improve the in vitro barrier model to make a more faithful replica of the actual placenta. To do this, we will isolate primary trophoblasts from human placenta and using novel technology we will create confluent barriers on transwell inserts, previously unfeasible due to the inability of trophoblasts to proliferate in vitro. These primary trophoblast barriers will be validated and optimised for NP exposures. Then, taking DNA damage, chromosomal aberrations and cytokine secretion as our endpoints the mechanisms of damaging signalling within the barrier in response to NP exposures will be investigated. In addition, imaging of NP uptake by the barrier and NP localisation within the cells will be performed.

Methodology/Skills associated with project:
Set up a human primary trophoblast in vitro model of the placenta. Validate this model using techniques such as immunofluorescence for cell junctions, TEER measurements, histological analysis and electron microscopy. Mechanisms and actions of signalling (role of connexins, pannexins and purinergic transmission) in response to nanoparticle exposures will be investigated within the barrier as well as the resultant damage in cells beneath the barrier. The effect of coating nanoparticles in different protein coronas or with select modifications to their surface chemistry will also be explored.
Project ESR8

Project Title: Design of degradable targeted nanoparticle-based delivery vehicles
Fellow’s Name: Marilena Hadjidemetriou
Tutor’s name: Prof. Kostas Kostarelos

Project Description:
The project is concerned with the design of degradable and targeted nanoparticle-based delivery vehicles with an emphasis on brain tissue. This will involve fabrication of different types of nanoparticles with different shapes and chemical consistencies, their characterisation, cell internalization kinetics, tissue distribution (in vivo) and degradation kinetics (in vitro and in vivo). The results will be exchanged with our European collaborators to develop nano-bio interfacial strategies for intracellular delivery beyond barrier crossing. The Early Stage Researcher will use and develop existing state of the art methods and materials in nanotechnology for cellular drug delivery. The work will be performed in close collaboration with members of the network and in particular the Institute for Molecular Medicine at the University of Southern Denmark (Denmark), the Helmholtz Centre for Infection Research (Germany) and the AvantiCell Science Ltd (UK).

Methodology/Skills associated with project:
Fabrication of different types of nanoparticles; physicochemical characterisation; cell internalization kinetics/cell culture; tissue distribution (in vivo) and degradation kinetics (in vitro and in vivo); optical microscopy; Epifluorescence microscopy.
Project ESR9

Project Title: Designing the bionanointerface for transcytosis and barrier crossing
Fellow’s Name: Luciana-Maria Herda
Tutor’s name: Prof. Kenneth A. Dawson (NUID UCD); Co-supervisor: Prof. Kostas Kostarelos (UNIMA)

Project Description:
This project aims to the design novel nanocarriers, and surface modifications, for controlled negotiation of biological barriers (mainly the blood-brain barrier). This project will also focus on the development of methods to control and reduce functional nanoparticle batch to batch variability. For this purpose, synthesis of functional probe nanoparticles incorporating features optimised for spectroscopic detection will be employed, together with investigation of surface functionalization chemistry to optimise for combined properties of dispersion stability, surface reactivity for biofunctionalization and overall product functionality in complex environments. Training in the design of biomimetic targeting moieties will also be included, so as to minimize non-specific biomolecular binding and to optimize receptor engagement for endocytosis.

As part of this Marie Curie program an emphasis will be placed upon collaborative aspects, with the fellow cooperating with other ESR/ERs across the project as the work builds. Research secondments at the partner and other institutes (UNIMA, SDU, Genzyme), will form part of the training experience while enhancing the overall output quality.

Methodology/Skills associated with project:
Wet-laboratory synthetic methods, characterised by a range of approaches including: light scattering, analytical centrifugation, UV-Vis and fluorescence spectroscopy, electron microscopy, NMR, mass spectrometry, measurements through cell biology methods.
Project ER1

Project Title: Cell-Based Nanosafety Assay Design and Validation for Accreditation and Dissemination
Fellow’s Name: Dr Maria Rita Fabbrizi
Tutor’s name: Dr Colin J Wilde (ACS)

Project Description:
The project will take lead cell-based systems with proven ability to report nanoparticle toxicity and translate them into robust assays capable of wide dissemination across partner and third-party laboratories. The objective shall be to validate the assay components and methodology to the extent that allows them to be submitted for formal accreditation. The assay translation process shall develop a standardised method for presenting standard nanoparticles (as positive controls) and for presentation of nano-objects captured from industrial materials using a membrane-based system; assay production shall also incorporate technology for cryopreserving cells in situ in multi-well plates, optimising reproducibility by minimising assay manipulations.

As part of this Marie Curie program, an emphasis will be placed upon collaborative aspects with other ESR/ERs across the project as the work builds. Research secondments at the partner and other institutes (NUID UCD/EPOS) will form part of the training experience while enhancing the overall output quality.

Methodology/Skills associated with project:
Set up and optimisation of in vitro renal slit diaphragm models; different imaging techniques, such as confocal- and fluorescence- microscopy, as well as other methods, to quantify nanoparticle localization and passage through barrier. Significant focus will be placed on gaining quantitative reproducible experimental data to characterise the detailed mechanism by which the nanoparticles are transported.
Project ER2

Project Title: Experimental testing of nanoparticulate systems for crossing intact and disrupted barriers.
Fellow’s Name: Dr Nikolaos Mandalos
Tutor’s name: Andreani Odysseos and Constantinos Pitris (EPOS)

Project Description:
Project aims to investigate the structure and function of diseased and intact (healthy) blood-brain barrier (BBB) in animal models, with Glioblastoma Multiforme (GM) oncogenic pathology. This study will examine the molecular and genetic mechanism that governs the development, maintenance, and homeostasis of BBB under normal and tumorigenic conditions. The novel perspective is to further develop and test nanoparticulate systems (NPs) that can efficiently cross the BBB, delivering effective drug therapy and early diagnosis against tumors.
The ultimate goal of this study is the identification of choreographic distribution of different nanoparticles in normal and diseased (GM) BBB in orthotopic mouse models and in tumor spheroids

Methodology/Skills associated with project:
Development of novel three-dimensional tumor spheroid scaffolds for the in vitro study of BBB within GM. Molecular analysis and characterization of the biological properties of three-dimensional spheroid tissue constructs with immunohistochemistry and hematoxylin and eosin (H&E) staining. Generation of orthotopic human GM tumor xenograft mouse models with intact and disrupted BBB. Efficacy of nanoparticles to cross the BBB will be tested by ATP-ase, proteinase assays, and visualization using optical, confocal and electron microscopy.