Final Report Summary - PATHCHOOSER (Innovative, mechanistic-based strategies for delivery of therapeutic macromolecules across cellular and biological barriers)
Nanomedicine offers us the capability to significantly change the course of treatment for life threatening diseases. Many of the most significant current therapeutic targets, to be viable in practice, require the efficient crossing of at least one biological barrier. However, the efficient and controlled crossing of the undamaged barrier is difficult. The range of small molecules that can successfully do so is limited in size and physiochemical properties, greatly restricting the therapeutic strategies that may be applied. In practice, after several decades of limited success, there is a broad consensus that new multidisciplinary, multi-sectoral strategies are required. Key needs include detailed design and understanding of the bionano-interface, re-assessment of in vitro models used to assess transport across barriers, and building regulatory considerations into the design phase of nanocarriers. The overarching premises of the PathChooser ITN are that: (i) significant advances can only be made by a more detailed mechanistic understanding of key fundamental endocytotic, transcytotic, and other cellular processes, especially biological barrier crossing; (ii) elucidating the mode of action / mechanism of successful delivery systems (beyond current level) will ensure more rapid regulatory and general acceptance of such medicines; (iii) inter-disciplinary knowledge from a range of scientific disciplines is required to launch a genuine attack on the therapeutic challenge. The PathChooser ITN program of research and training has equipped the next generation of translational scientists with the tools to develop therapies for a range of currently intractable and economically unviable diseases.
WP1 - Novel Nanocarriers Design, Development, Synthesis
From the synthesis and development point of view, a set of model silica nanocarriers has been developed by Fellow Luciana Herda, UCD, with precisely controlled properties and variable surface functionalization. A range of PEG ligand architectures and protein (Transferrin (Tf) and Apolipoprotein E (ApoE) isoforms) surface coverage has been employed, in an effort to understand and elucidate the impact of surface architecture on the presentation of targeting moieties and, potentially, nanoparticle functionality in biological systems. Since there are few relevant characterization methodologies to elucidate molecular arrangement at the nanoparticle surface, the implementation of an immunomapping approach to label relevant epitopes within the structure of surface proteins has been achieved. This approach offers relevant information on their presentation upon grafting to particles and assists in nanoparticle design.
The work of Fellow Martina Tuttolomondo, SDU, is described in the following manuscript that was featured on the cover of the journal Molecular Therapy – Nucleic Acids: Martina Tuttolomondo, Cinzia Casella, Pernille Lund Hansen, Ester Polo, Luciana M. Herda, Kenneth A. Dawson, Henrik J. Ditzel and Jan Mollenhauer, Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery, In Molecular Therapy - Nucleic Acids, Volume 8, 2017, Pages 264-276, ISSN 2162-2531, https://doi.org/10.1016/j.omtn.2017.06.020. Meanwhile, Fellow Marilena Hadjidemetriou from the University of Manchester has written a News & Views article about the protein corona concept in Nature Nanotechnology.
WP2 - Mechanisms of uptake of biomacromolecules across cellular and non-cellular barriers
The main aim of WP2 was to clarify endo- and exocytosis processes and to develop and validate cell-based barrier platforms, such as in vitro models, to address the biokinetics and the uptake of nanoparticles of different sizes or surface chemistry. The fellows involved in this WP have addressed these questions directly.
Diána Hudecz from UCD, has developed an in vitro model of the Blood-Brain Barrier (BBB) for live cell imaging using an ultrathin silicon nitride membrane, which enables the user to study cellular mechanisms on nanoparticle trafficking in endothelial cells and astrocytes. Xabier Murgia from HZI, has developed a Transwell-based in vitro model of the airways comprising epithelial cells and human tracheal mucus, which can be used to study the uptake of nanoparticles in the presence of mucus, but can also be applied for permeation studies with small drug molecules. Prasath Paramasivam from MPI, has performed a new pilot screen and identified small interfering RNA (siRNA) delivery enhancers in mouse hepatocytes. This information will be used to redesign siRNAs and to develop drug delivery systems.
WP3 - Mechanisms of uptake of biomacromolecules across biological tissue barriers
At ACS Maria Fabbrizi undertook a project to develop novel cell models for the study of renal filtration. The objective was to create a tool for preclinical testing of nano-object processing in the kidney. The project succeeded in isolating and characterising primary human podocytes from tissue obtained as surgical excess with full ethical permission, and also investigated the potential of cells from an iPS progenitor to achieve a renal phenotype and offer a renewable alternative to primary-cell supply. The trainee gained skills in the development of three-dimensional (3D) cell models, and acquired insight into the dynamics and business context of commercially-orientated R&D.
Meanwhile at Bristol, a novel model for the placental barrier was developed by fellow Catherine Gilmore. The placenta is one of the least understood human organs, and yet it is one of the most important, supporting fetal growth and development throughout pregnancy. Placental malfunction has been linked to many of the most common complications of pregnancy, such as miscarriage and preeclampsia, and in recent years, a role for the placenta in the fetal programming of adult disease has also been uncovered. Previous work in Dr. Patrick Case’s laboratory has demonstrated that the placental barrier can signal damage to fetal cells in response to exposures, such as hypoxic insults or benzene metabolites, with potentially lifelong implications for the offspring. For these reasons, it is imperative that our understanding of the placenta and its influence on fetal health has been improved.
To achieve this, a novel placental barrier model, using primary human trophoblasts, was constructed by coating the cells with extra-cellular matrix components. The model has many structural and molecular similarities to the in vivo placental barrier, including the ability to syncytialise. Adaptations to the model were also made to incorporate more of the complexity of the in vivo situation; a preeclamptic placental barrier model was produced, and a model that mimics the first trimester placental barrier structure. A vascularised placental barrier model was also established in which a HUVEC vascular network within a multi-layered fibroblast tissue structure was constructed beneath the placental barrier model, to replicate the feto-placental vasculature.
This novel model was used to examine how placental barrier signalling in response to uterine exposures could impact upon feto-placental vasculature. Benzene exposure and certain hypoxic insults were found to elicit a placental barrier signalling response that indirectly affected HUVEC angiogenesis. This suggests that the placental barrier response to its environment can impact upon placental vascular development, with potentially negative consequences for fetal health and development. As a result of this work, a novel model has been developed which is not only applicable to nanoparticle research but also to the study a broad number of biological questions.
At KCL, Fellow Anne Iltzsche’s project was entitled “Apolipoprotein E and A targeted albumin nanoparticle delivery to the CNS: Mechanism and route of delivery”. The project succeeded in demonstrating that ApoE targeted 200 nm nanoparticles across the BBB in a dose-dependent manner via apolipoprotein receptors. Within 5 mins after intravenous administration these particles were distributed within endothelial cells of the BBB, 15 mins later they were within the cytoplasm of astroglia and neurons and within 30 mins they were widely distributed throughout the brain. The project has successfully demonstrated that this apolipoprotein dependent transcytotic mechanism is capable of delivering potential drug cargo widely within the central nervous system thus forming the basis of a generic and novel drug delivery system.
WP4 - Route, organ and tissue specific delivery strategies
One possible use of nanoparticles in nanomedicine is as drug carriers for selectively targeting cancer cells. In the case of the brain tumours, before these materials can encounter cancer cells they will need to penetrate the BBB. But even if this process can be accurately modelled, a reliable and validated computational model of the growth of a brain tumour must be implemented so the effect of the nanoparticles on the tumour cell can be evaluated. Under this work package, fellows from EPOS, Nikolas Mandalos and Savvas Savva, have worked to create a new model to study brain tumour development.
WP5 - Implementation of Fellows Training
The Training Pathway of the PathChooser ITN has comprised of an outstanding achievement in both training and network as witnessed by individual Fellows’ accomplishments and network-wide events. Each one of the ten Early Stage Researchers and two Experienced Researchers has demonstrated diligence, determination and ambition for excellence in the sciences. This has been facilitated by the respective institutions and supervisors, the encouragement to access state of the art learning and development environments, including training in novel and unique techniques, and beyond, to include consideration of core transferable skills. The fellows have benefited in many cases therefore from well-developed host laboratories where there are many other activities enabling them to address exceptionally complex tasks, as well as learning the skills to link into and harness the surroundings in a creative manner. This groundwork and foundation in knowledge has helped the Fellows to increasingly benefit from their research visits to other laboratories.
Many other activities involving broader collaboration, dissemination, outreach and public engagement – some being awarded with prestigious prizes and acknowledgements – have duly served the Commission’s goals. As expected, and hoped, in a Marie Curie Program, the fellows both individually and collectively have developed a distinctive personal ethos, and esprit de corps. The project’s pride, however, resides in the generation of a kernel of PhD holders, most of whom have already excelled in their career development in both academia and industry with the acquisition of top-level scientific positions in a highly competitive global environment.
Further details of the project can be found on our website: https://www.pathchooser.eu/
WP1 - Novel Nanocarriers Design, Development, Synthesis
From the synthesis and development point of view, a set of model silica nanocarriers has been developed by Fellow Luciana Herda, UCD, with precisely controlled properties and variable surface functionalization. A range of PEG ligand architectures and protein (Transferrin (Tf) and Apolipoprotein E (ApoE) isoforms) surface coverage has been employed, in an effort to understand and elucidate the impact of surface architecture on the presentation of targeting moieties and, potentially, nanoparticle functionality in biological systems. Since there are few relevant characterization methodologies to elucidate molecular arrangement at the nanoparticle surface, the implementation of an immunomapping approach to label relevant epitopes within the structure of surface proteins has been achieved. This approach offers relevant information on their presentation upon grafting to particles and assists in nanoparticle design.
The work of Fellow Martina Tuttolomondo, SDU, is described in the following manuscript that was featured on the cover of the journal Molecular Therapy – Nucleic Acids: Martina Tuttolomondo, Cinzia Casella, Pernille Lund Hansen, Ester Polo, Luciana M. Herda, Kenneth A. Dawson, Henrik J. Ditzel and Jan Mollenhauer, Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery, In Molecular Therapy - Nucleic Acids, Volume 8, 2017, Pages 264-276, ISSN 2162-2531, https://doi.org/10.1016/j.omtn.2017.06.020. Meanwhile, Fellow Marilena Hadjidemetriou from the University of Manchester has written a News & Views article about the protein corona concept in Nature Nanotechnology.
WP2 - Mechanisms of uptake of biomacromolecules across cellular and non-cellular barriers
The main aim of WP2 was to clarify endo- and exocytosis processes and to develop and validate cell-based barrier platforms, such as in vitro models, to address the biokinetics and the uptake of nanoparticles of different sizes or surface chemistry. The fellows involved in this WP have addressed these questions directly.
Diána Hudecz from UCD, has developed an in vitro model of the Blood-Brain Barrier (BBB) for live cell imaging using an ultrathin silicon nitride membrane, which enables the user to study cellular mechanisms on nanoparticle trafficking in endothelial cells and astrocytes. Xabier Murgia from HZI, has developed a Transwell-based in vitro model of the airways comprising epithelial cells and human tracheal mucus, which can be used to study the uptake of nanoparticles in the presence of mucus, but can also be applied for permeation studies with small drug molecules. Prasath Paramasivam from MPI, has performed a new pilot screen and identified small interfering RNA (siRNA) delivery enhancers in mouse hepatocytes. This information will be used to redesign siRNAs and to develop drug delivery systems.
WP3 - Mechanisms of uptake of biomacromolecules across biological tissue barriers
At ACS Maria Fabbrizi undertook a project to develop novel cell models for the study of renal filtration. The objective was to create a tool for preclinical testing of nano-object processing in the kidney. The project succeeded in isolating and characterising primary human podocytes from tissue obtained as surgical excess with full ethical permission, and also investigated the potential of cells from an iPS progenitor to achieve a renal phenotype and offer a renewable alternative to primary-cell supply. The trainee gained skills in the development of three-dimensional (3D) cell models, and acquired insight into the dynamics and business context of commercially-orientated R&D.
Meanwhile at Bristol, a novel model for the placental barrier was developed by fellow Catherine Gilmore. The placenta is one of the least understood human organs, and yet it is one of the most important, supporting fetal growth and development throughout pregnancy. Placental malfunction has been linked to many of the most common complications of pregnancy, such as miscarriage and preeclampsia, and in recent years, a role for the placenta in the fetal programming of adult disease has also been uncovered. Previous work in Dr. Patrick Case’s laboratory has demonstrated that the placental barrier can signal damage to fetal cells in response to exposures, such as hypoxic insults or benzene metabolites, with potentially lifelong implications for the offspring. For these reasons, it is imperative that our understanding of the placenta and its influence on fetal health has been improved.
To achieve this, a novel placental barrier model, using primary human trophoblasts, was constructed by coating the cells with extra-cellular matrix components. The model has many structural and molecular similarities to the in vivo placental barrier, including the ability to syncytialise. Adaptations to the model were also made to incorporate more of the complexity of the in vivo situation; a preeclamptic placental barrier model was produced, and a model that mimics the first trimester placental barrier structure. A vascularised placental barrier model was also established in which a HUVEC vascular network within a multi-layered fibroblast tissue structure was constructed beneath the placental barrier model, to replicate the feto-placental vasculature.
This novel model was used to examine how placental barrier signalling in response to uterine exposures could impact upon feto-placental vasculature. Benzene exposure and certain hypoxic insults were found to elicit a placental barrier signalling response that indirectly affected HUVEC angiogenesis. This suggests that the placental barrier response to its environment can impact upon placental vascular development, with potentially negative consequences for fetal health and development. As a result of this work, a novel model has been developed which is not only applicable to nanoparticle research but also to the study a broad number of biological questions.
At KCL, Fellow Anne Iltzsche’s project was entitled “Apolipoprotein E and A targeted albumin nanoparticle delivery to the CNS: Mechanism and route of delivery”. The project succeeded in demonstrating that ApoE targeted 200 nm nanoparticles across the BBB in a dose-dependent manner via apolipoprotein receptors. Within 5 mins after intravenous administration these particles were distributed within endothelial cells of the BBB, 15 mins later they were within the cytoplasm of astroglia and neurons and within 30 mins they were widely distributed throughout the brain. The project has successfully demonstrated that this apolipoprotein dependent transcytotic mechanism is capable of delivering potential drug cargo widely within the central nervous system thus forming the basis of a generic and novel drug delivery system.
WP4 - Route, organ and tissue specific delivery strategies
One possible use of nanoparticles in nanomedicine is as drug carriers for selectively targeting cancer cells. In the case of the brain tumours, before these materials can encounter cancer cells they will need to penetrate the BBB. But even if this process can be accurately modelled, a reliable and validated computational model of the growth of a brain tumour must be implemented so the effect of the nanoparticles on the tumour cell can be evaluated. Under this work package, fellows from EPOS, Nikolas Mandalos and Savvas Savva, have worked to create a new model to study brain tumour development.
WP5 - Implementation of Fellows Training
The Training Pathway of the PathChooser ITN has comprised of an outstanding achievement in both training and network as witnessed by individual Fellows’ accomplishments and network-wide events. Each one of the ten Early Stage Researchers and two Experienced Researchers has demonstrated diligence, determination and ambition for excellence in the sciences. This has been facilitated by the respective institutions and supervisors, the encouragement to access state of the art learning and development environments, including training in novel and unique techniques, and beyond, to include consideration of core transferable skills. The fellows have benefited in many cases therefore from well-developed host laboratories where there are many other activities enabling them to address exceptionally complex tasks, as well as learning the skills to link into and harness the surroundings in a creative manner. This groundwork and foundation in knowledge has helped the Fellows to increasingly benefit from their research visits to other laboratories.
Many other activities involving broader collaboration, dissemination, outreach and public engagement – some being awarded with prestigious prizes and acknowledgements – have duly served the Commission’s goals. As expected, and hoped, in a Marie Curie Program, the fellows both individually and collectively have developed a distinctive personal ethos, and esprit de corps. The project’s pride, however, resides in the generation of a kernel of PhD holders, most of whom have already excelled in their career development in both academia and industry with the acquisition of top-level scientific positions in a highly competitive global environment.
Further details of the project can be found on our website: https://www.pathchooser.eu/