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SEVENTH FRAMEWORK PROGRAMMETHE PEOPLE PROGRAMME

Annex I - “Description of Work”*

PART A:

Grant agreement for: Initial Training NetworksCall identifier: FP7-PEOPLE-2013-ITN

Implementation mode: Multi-ITN

Project acronym: PathChooserGrant agreement no.: 608373

Project full title: Innovative, mechanistic-based strategies for delivery of therapeutic macromolecules across cellular and biological barriers

Date of approval of Annex I by REA: 10/07/2013Project start date: 1st day of the month after the last signature of the GA parties

* This Annex I refers to the 2013 PEOPLE Work Programme (European Commission C(2012)4561 of 9 July 2012)

Ref. Ares(2013)2831470 - 05/08/2013

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A.1 Project abstract

Keywords: bionano-interface, biological barriers, therapeutics, regulation, entrepreneurial, training, mobility, inter-sectoral, multi-disciplinary, nanocarriers

Abstract:Nanomedicine offers 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 (via diffusive or other non-specific processes) 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 multi-disciplinary, multi-sectoral strategies are required. Key needs include detailed design and understanding of the bionano-interafce, 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. Paramount in this is the design and characterization of the in situ interface between the carrier system and the uptake and signalling machinery. (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 will equip the next generation of translational scientists with the tools to develop therapies for a range of currently intractable (e.g. hidden in the brain) and economically unviable diseases (e.g. orphan diseases affecting a limited population).

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PART B:B.1 List of participants

B.1.1. List of Participants (full beneficiaries)

Beneficiary Number * Beneficiary short name**

Private Sector (Y/N)

SME (Y/N)

Country Month enterproject***

Month exit project

1 National University of Ireland, University College Dublin

NUID UCD N N Ireland 1 48

2. SYDDANSK UNIVERSITET

SDU N N Denmark 1 48

3 HELMHOLTZ-ZENTRUM FUER INFEKTIONSFORSCHUNG GMBH

HZI N N Germany 1 48

4 KING'S COLLEGE LONDON

KCL N N United Kingdom

1 48

5 E.P.O.S. IASIS RESEARCH AND DEVELOPMENT LTD

EPOS IASIS

Y Y Cyprus 1 48

6 MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.

MPI-CBG N N Germany 1 48

7 UNIVERSITY OF BRISTOL

UNIVBRIS N N United Kingdom

1 48

8 THE UNIVERSITY OF MANCHESTER UNIMA N N United

Kingdom1 48

9 AvantiCell Science Ltd ACS Y Y United

Kingdom1 48

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B.1.2. List of Associated Partners (including role and status)

N° Associated Partner name

Short name Country Organisation type*

SME (Y/N)

Role in the project**

1 Genzyme, a Sanofi Company

Genzyme United States of America

Private:

Rare Diseases Division

Y SEC: Transferable skills training, potential secondment opportunities (at Sanofi, France).

2 University of Cyprus Business Incubator, DIOGENES

UCY-DIOGENES

Cyprus Private: Business Incubator

Y TR: Courses in technology transfer, entrepreneurship development

Genzyme is one of the world’s leading biotechnology companies. Its approximately 10,000 employees work in countries throughout the world and are united by a common goal: to make a major positive impact on the lives of people with debilitating diseases. Since its founding in 1981, Genzyme has grown from a small start-up to a diversified enterprise with 2010 revenues of $4.05 billion. Over the past three decades, Genzyme has introduced a number of breakthrough treatments in several areas of medicine, which have provided hope to patients who previously had no viable treatment options.Genzyme’s products are focused on rare genetic diseases, multiple sclerosis, cardiovascular disease and endocrinology. The company’s commitment to innovation continues today with a substantial research and development program focused on these fields, as well as other areas of unmet medical need.Key Persons and Expertise: Dr. Seng H. Cheng (Head of Research and Early Development, Rare Diseases Science)

Diogenes Business Incubator University of Cyprus, wholly owned by the University of Cyprus, Cyprus’ largest public University, is a high technology business incubator aims to commercialize R&D results by creating business value and is geared to pioneering the transformation of Cyprus into an important center in the Eastern Mediterranean in the area of commercializing high technology research and innovative ideas and is committed to developing entrepreneurship in the country as an important component of the process. Member of the Incubator Forum, the Cyprus Association of Business Incubators, the National Business Incubator Association (USA), the UK Business Incubator Association and the Cyprus Chamber of Commerce and is in close collaboration with the Ministry of Commerce Industry and Tourism and a founding member of the Cyprus MANUFUTURE Platform. Diogenes experience lies in entrepreneurship development, commercialization of R&D results and creating sustainable start-up companies that are enhanced with the Incubator’s network. Diogenes concentrates on commercializing R&D results that demonstrate prospects of market viability and success and offers supportive and strategic value adding services and pre and post incubation strategic services, facilitating the smooth transition of R&D results into commercialization and a successful start-up. Diogenes is part of the organizing committee and a mentor of the Cyprus Entrepreneurship Competition. Additionally, Diogenes supports Open Coffee Cyprus series of events through the provision of entrepreneurship and start-up related courses / presentations to the participants. Diogenes has extensive experience in mentoring and coaching of researchers and young entrepreneurs, in matters related to entrepreneurship and startup support. Teaching expertise/ materials: Entrepreneurship Development, Strategy & Business Planning, Start-up basics, Networking, Pitching etc.Key Persons and Expertise: Stavriana A. Kofteros, Supervisor/lead trainer/coach/mentor. Prof. Skevos Evripidou, computer scientist. Chairs the organization committee of the Cyprus Entrepreneurship Competition. Extensive experience in technology entrepreneurship

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B.2 S&T Quality

B.2.1. Objectives of the research programme

Safe crossing of biological barriers would open the way to therapeutics for many intractable diseases, and lower the cost of treating many others. The overarching intention of PathChooser is to create the knowledge (and develop the people to further build that knowledge) to understand how to control the bio-nano interface to promote biological barrier crossing, for biologically and cinically‘relevant’situations, including particularly complex biological milieu.1. Establish the mechanistic foundations as a basis for a coherent program and collaboration –

we seek to build a generally applicable set of platforms (in vitro and in vivo models and associated tools), tailored to different barriers and therapeutic targets. (ESR1, ESR2, ESR3, ESR4, ESR5, ESR7, ER1, ER2)

2. Overcome the key challenges in targeted delivery involving targets beyond Biological Barriers - by harnessing natural endogenous vesicular processes such endo-, exo- and transcytosis as the foundation of delivery across the cellular barriers, rather than simply encapsulating therapeutics to protect them and to reduce their toxicity. (ESR1, ESR3, ESR5, ESR6, ESR7, ESR9, ER1, ER2)

3. Develop nano-bio interfacial strategies for intracellular delivery beyond barrier crossing -since transport across barriers must be accomplished in a complex biological milieu that can significantly modify the nature of the carrier-system interface, strategies to optimise the presentation of the bio-nano interface and overcome non-specific binding are needed. (ESR2, ESR6, ESR8, ESP9, ER1, ER2)

4. Synthesise the data into mechanistic knowledge regarding the biodistribution of nanomedicines that allows translation into clinical settings - detailed mechanistic knowledge regarding their uptake, biodistribution, clearance and/or in situ degradation will be generated which will enable efficacy and safety to be assessed in parallel for future nanomedicines. (ESR1, ESR3, ESR4, ESR5, ESR7, ER1, ER2).

All PathChooser Fellow’s projects contribute to the training objectives. The added value consists of each project and partner contributing platforms and model systems, which allows setting benchmarks and cross-testing of the delivery systems in a high number of different settings.

Table 2.1.1 List of Work Packages

Work package No*

WP Type** Work package title Lead beneficiary

Start month

End month

1 RTD Novel Nanocarriers Design, Development, Synthesis

SDU 7 42

2 RTD Mechanisms of uptake of biomacromolecules across cellular and non-cellular barriers

HZI 7 42

3 RTD Mechanisms of uptake of biomacromolecules across biological tissue barriers

KCL 7 42

4 RTD Route, organ and tissue specific delivery strategies

NUID UCD 7 42

5 TR Implementation of the training pathway EPOS-IASIS 1 486 DISS Dissemination NUID UCD 1 487 MGT Management NUID UCD 1 48

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Table 2.1.2 List of Milestones List of Milestones

Work Package

MilestoneNo.

Milestone Lead Beneficiary

Month

1-4 1 Yearly and Mid-term scientific progress reports of all fellows & associated scientific deliverables

1, with 2-12 18, 24, 30, 42

1-4 2 Yearly report on exchange of fellows 1, with 2-12 18, 30, 421-4 3 Periodical report about seminar talks given by

fellows1, with 2-12 12, 18, 24, 30,

36, 421-4 4 Yearly report on congress activities of fellows 1, with 2-12 18, 30, 421-4 5 Final scientific milestone: All PhD theses

completed1, with 2-12 48

5 6 Fellows are selected and appointed All 65 7 Periodic report of supervisors about fellows’

proficiency1, with 2-12 12, 18, 24, 30,

36, 425 8 Periodic report of supervisors about fellows’

rotations 1, with 2-12 12, 24, 36

5 9 Periodic report of supervisors about fellows’ participation in journal clubs and lectures

1, with 2-12 12, 18, 24, 30, 36, 42

5 10 Completion of graduate courses required by host universities/institutions

1, with 2-12 30

5 11 Periodic report of supervisors about fellows’ participation in training courses

1, with 2-12 18, 30, 42

5 12 Mid-term and final evaluation of the training programme

1, with 2-12 24, 48

Table 2.1.3 Deliverables List

List of Deliverables

WP Deliver. No.

Deliverable Lead Beneficiary

Nature Dissemination Month

1 1.1 Relevant properties of NPs to engage with endogenous processes defined

UNIMA R PU 18

1 1.2 Validation of use of endogenous processes by model carrier particles

SDU R PU 24

1 1.3 Outline of expected degradation and clearance outcomes of NP carriers.

UNIMA R PU 24

1 1.4 Synthesis and characterization of chemical purity of carriers, as well as embedded materials.

UNIMA O, R PU 30

1 1.5 In depth physiochemical characterization of all carriers in situ prior to and during uptake and final sub-cellular localisation / following transcytosis.

NUID UCD R PU 30

1 1.6 Redesigned nano-carriers according to a basic parameter set (size, shape, surface, bionanointerface).

SDU O, R PU 36

2 2.1 Characterization of cellular functions (cell lines, primaries, endothelia and epithelia restored functions –transcytosis etc.).

HZI R PU 18

2 2.2 Reconstruction of relevant barrier components and assessment of nanocarrier interactions

HZI O, R RE 24

2 2.3 Standardised biological fluid environment to ensure representative ‘protein corona’ around particles.

NUID UCD O, R PU 24

2 2.4 Time-resolved intra-barrier nanoparticle distributions in presence of stress or other environmental conditions.

ACS R PU 30

2 2.5 Analytical tools for tracking & extraction of NPs along uptake/translocation pathways.

MPG R RE 30

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3 3.1 Cross-platform protocols in vivo dosing KCL R RE (PU in time) 183 3.2 Protocols for primary cells and tissue

extractionUNIVBRIS O, R RE 18

3 3.3 Classification and analysis of relevant (species dependent) mucous

KCL R PU 36

4 4.1 Creation of data-basing tools NUID UCD O, R PU 184 4.2 Database of characterized endogenous

pathways in cells and biological barriersMPG R RE 24

4 4.3 Computational modelling of barrier crossing

EPOS R PU 36

5 5.1– 5.6 Periodical scientific and collaboration (secondments) progress reports

NUID UCD, All

R RE 18, 30, 42

5 5.7–5 .15

Periodical reports of fellows’ seminar and congress presentations / participation

NUID UCD, All

R RE 10, 16, 22, 28, 34, 40

5 5.16 Fellows’ PhD thesis All O PU 485 5.17 Report of fellows selection and

appointmentNUID UCD, All

R RE 6

5 5.18–5.32

Periodical reports of fellows’ proficiency, rotations, participation in journal clubs

NUID UCD, All

R RE 12, 18, 24, 30, 36, 42

5 5.33 Report of fellows completion of graduate courses

NUID UCD, All

R RE 28

5 5.34-5.36

Periodical reports of fellows participation to training courses and activities

NUID UCD, All

R RE 15, 24, 33

5 5.37-5.38

Mid-term and final reports of PathChooser research & training outcomes

NUID UCD, All

R PU 24, 48

6 6.1 Development of a website All Diss 66 6.2 Report on the Mechanisms of

nanoparticle passage across biological barriers. All fellows will disseminate their results at this conference

KCL, All Diss 18

6 6.3 Report on Meet-the-Manager Workshop. All fellows are required to take part

All Diss 30

6 6.4 Report on the Therapeutic strategies involving nanoparticles actively transporting across biological barriers International conference on. All fellows will disseminate their results at this conference

EPOS, All Diss 36

B.2.2. Research methodology and approach

To efficiently pass through a biological barrier an engineered nanocarrier should:

(1) provide protection against biomolecular degradation: In principle the task of protecting a biomolecule is easily accomplished by embedding it within a carrier construct, the primary challenges being of a technical nature to ensure high loading and maintained function. Relevant questions (related to biodistribution, mechanisms of accumulation and clearance) regarding the degradative pathways, and the mechanisms and side products of the nanoparticle carrier will be addressed, from a regulatory viewpoint.

(2) limit basic immunological responses and evade primary clearance mechanisms: In principle the size range of less than a couple of hundred nanometers can lead to muted primary immunological responses, provided the in situ nanocarrier interface (surface, including grafted molecules, and adsorbed biomolecules) with the biological environment is controlled. Efforts will include designing carriers with size, shape and surface characteristics, building on biomimetic analogies with naturally occurring stealth mechanisms (e.g. those used by pathogens to escape immune responses or create an anti-inflammatory environment).

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(3) survive presence of shear, flow, non-steady microstress environments: For different barriers some information is known about the flows, flow rates, local shear and other mechanical/viscoelastic phenomena within which transport occurs. These parameters are critical for determining on-/off-rates and this will be incorporated into the experimental design, and in the evolution of specific biological barrier models.

(4) efficiently traverse mucous and/or extracellular matrix: Carrier size, shape, deformability and interaction with the in situ interface significantly affect the transport across these passive materials barriers, and evidence suggests that (for many cases) this determines the efficiency of delivery. In order to decipher these determinants of delivery we will build on natural mucoadhesive mechanisms.

(5) discriminate between a wide range of potential receptors in the endothelium (and other organs) in the presence of non-specific adhesions: Biological barriers, because they are designed to transport appropriate molecules have high surface areas (e.g. 20 m2 blood-brain, 70 m2 lung, 300 m2 gut), so in principle the cross section for transmission of nanoparticles by active processes is large. However, there are competing factors in successful crossing, for example from non-specific adhesion or unplanned incorporation into the endothelium, which limit the efficiency of crossing the target barrier. This training program emphasizes the discrimination (or specificity) of the targeting nanoparticles to differentiate between what is taken across the correct barrier by the desired process, from uptake by all other routes (possibly leading to accumulation in the wrong target), and will provide analytical and screening tools to ensure discrimination at early stages of carrier design.

(6) traverse the relevant endothelial barrier, without significant accumulation in the endothelium itself: Entry into cells of the epithelium or endothelium does not guarantee its successful crossing (for example by transcytosis) for the default pathway is often endolysosomal, as for most nanoparticle-cell–level interactions. In effect, one must exploit the intracellular sorting machinery to pick up a basal segment of the pathway, rather than the typical destination of late endosomes, and lysosomes. Nanocarriers that fail to do this constitute not just a loss of efficiency in therapeutic load, but also a potentially subtle form of toxicity. On the other hand, specific decisions can be made to use these accumulation of drugs within the endothelium (carefully managed), or paracrine signalling across the barrier can be promoted. Any of these mechanisms, can be efficacious, if understood and controlled.

(7) limit accumulation in the hepatic/reticuloendothelial system and manage renal clearance:Avoiding clearance processes for as long as possible allows more opportunity for targeting, and lowers the exposure dose required, and thereby the potential collateral damage to the reticuloendothelial system. Size, surface, shape, deformability, and accumulated proteins on the surface of the nanosystems affect this outcome. The lowering of non-specific protein binding by polymeric grafting has not been a sufficient response to this challenge, in part because scavenger receptors can continue to misdirect accumulation. New ways of managing the nanoparticle interface are incorporated into thisprogram, including e.g. a directed modulation of the adsorbed protein coat to improve and target delivery, and timely biodegradation.

Figure 1. Schematic of the biological barriers that will be the focus of the Path-Maker project.

Figure 2. Left: Schematic representation of the bionanointerface, and how this determines engagement of nanoparticles with biological interfaces via competitive interactions. Right:Effect of bionanointerface on particle

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uptake, relative to nanoparticle uptake in the absence of biomolecules.

Because current approaches cannot now be expected to radically ‘game change’, we have chosen to build on quite unique and innovative approaches, though in each case very significant preliminary scientific data exists for these. Each of the fellows projects maps onto one (or more) or the four scientific objectives listed in Section B2.1 and onto one (or more) of the research Work packages (WPs, WP1-4) given in Table 1 above.

Originality and innovative aspects of the research training programme

Overarching the entire project is the desire to use endogeneous pathways, rather than disrupting cellular processes (open tight junctions, crude cationic lysis of endosomes and lysosomes etc.) and damaging cells.

Approaches being investigated involve advanced conceptions of escaping the endo-lysosomal pathway based on exosomal biomimetics, rather than the classical breaching of late endosomes by cationic species. Several of the projects will exploring the role of local curvature on the trafficking pathways and entry into cells. This paradigm is suggested also by the roles of several bacterial and other organism derived toxins (for example, Shiga I) where non-classical (and sometimes non-receptor-mediated) pathways are highly successful in passing endothelia or evading lysosomal capture. The use of these biomimetics also suggests a strong role for certain lipids, in combination with prescribed membrane curvature. Indeed, it is also possible that higher axial ratio materials may enter by novel pathways, not previously appreciated.

The European Technology Platform NanoMedicine “Roadmaps in Nanomedicine: towards 2020” highlights the need for radical Innovation nanomedicines1, stating that concepts under evaluation must be translatable into the clinic under current or future regulatory environments, and such an evaluation should be done very early in the process (pre-funding or early project stage) and with the support of a company or clinical research group experienced in this arena. The PathChooser ITN takes this a step further towards realisation, by including such considerations and training as part of the core skills of the research training programme.

We anticipate three main employment fields for the fellows undergoing training within the PathChooser ITN, and the training content has been developed to address each of these needs, and develop a cohort of researchers with a good awareness of the challenges faced by their future colleagues in each of these arenas:

1. Academic research, which faces exciting challenges in translating the promise of nanomedicine into a rigorous mechanistic science, by designing the bio-nano-interface to allow optimal interactions with cellular barriers in vitro and in vivo.

2. Industry, which needs new methods to design the surface of nanocarriers, such that they effectively reach their targets, enabling delivery of key therapeutic molecules in lower doses, and with less side-effects, retaining therapeutic efficacy, and being effectively cleared by the body following delivery of the therapeutic load. A range of “plug-and-play” delivery motifs would enable industry to reduce research costs, and would thus enable them to provide more cost-effective medicines to patients who need them urgently.

3. Regulatory agencies, which rely on science-based advice when regulating nanomedicines, and who need to be certain that the models are realistic representation of in vivo, especially as society tends more and more away from animal testing (e.g. cosmetics are no longer tested on animals, even those that contain nanomaterials in their formulation). Non-technical communication skills will be critical here.

1http://www.etp-nanomedicine.eu/public/press-documents/publications/etpn-publications/091022_ETPN_ Report_2009 .pdf

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B.3 Training

B.3.1. Quality of the training programme

Various scientific disciplines will converge in the PathChooser project in order to train the professional nanomedical drug-delivery scientist. The future expert in nanocarriers for drug delivery should be skilled in chemistry, physics, biology, material sciences, medical sciences, engineering and nanotechnology in its strictest sense, but should also obtain knowledge in other areas, including design and implementation of translational research, clinical trials, industrial research, economics, communication of science, intellectual property and ethics. Such training is best achieved within the interdisciplinary and inter-sectoral environment of an ITN. The specific training activities aimed at developing nanointerfacial and nano-drug delivery expertise are the objective of WP5

Training-by-research. This will take place during the entire period of the internship of the fellows in the hosting institutions, under the supervision of expert researchers acting as tutors. The scientific topics and issues that will be addressed as part of the training course are described in the tasks and deliverables of WP1-4 and in the specific projects 1-11. Periodic progress reports will be checkpoints to assess the fellows’ advancements, both from the scientific point of view (Milestone 1 (M1): Scientific progress reports; see section B.4 Work plan) and in regard to acquisition of complementary skills such as communication, critical evaluation, problem-solving (M7: Fellows’ proficiency checkpoints). The lead scientist of each partner is responsible for these regular progress checks and for the provision of host-specific and fellow-specific training.

All fellows (ESR and ER) will be exposed to the ideas of good laboratory practice (GLP) in their projects-supported by industrial partners EPOS-IASIS, ACS, Genzyme etc. Interdisciplinary training is a key element of the PathChooser training that needs to encompass expertise in very different fields (chemistry, physics, biology, clinical sciences etc.). Secondments are specified in each individual project. Each ESR will spend an indicitive 3-6 months and ER at least 3 months during the project in one or more partner institutions, and all fellows will have professional interactions both with academic and industrial partners to ensure intersectoral exchange (M2: Collaboration progress reports; M8: Fellows’ rotations). Fellows will gain knowledge outside of natural sciences and engineering also by participating in obligatory course

s on Complementary Skills that will address key issues such as designing clinical trials, nanomedical regulatory procedures, project management, intellectual property right protection and industrial exploitation, and ethics in science (see Table B.3.1.2).

Progress of the programme will be monitored through the milestones, and its success through the deliverables. Management Committee and Supervisory Board will oversee this process, which the coordinator will summarise in comprehensive mid-term and final reports to the EU Commission

Training MilestonesM6. Fellows’ selection. Fellows will be selected after public calls and interviews, and ESR will be enrolled as graduate students into institutional PhD/specialisation courses following a public seminar. D5.17: Report of fellows selection and appointment (Mo 6).

M7. Proficiency check-points. Every six months a check-point will be met by each supervisor to evaluate fellows’ proficiency on overall training activities. In particular the supervisor will evaluate the fellows’ capacity to learn and improve their technical, manual and intellectual skills, verify their capacity to report and explain project data, and also the acquisition of complementary skills such as communication, language skills, computer skills, project management, ethics, IPR protection, team building, training on specialised instruments/equipment, etc. D5.18-23: Periodical reports of fellows’ proficiency (Mo 12, 18, 24, 30, 36, 42).

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M8. Fellows’ rotations. All fellows will be required to rotate to one or two of the other consortium institutions, to gain technical and cross-disciplinary expertise, and as part of collaborative efforts. These activities will be reported during the progress meetings. D5.24-26: Periodical reports of fellows’ rotations (Mo 12, 24, 36).

M9. Participation to journal clubs and lectures. Fellows are required to participate to journal clubs and lectures at their institutions, to learn presentation skills and critical capacity. D5.27-32: Periodical reports of fellows’ participation to journal clubs and lectures (Mo 12, 18, 24, 30, 36, 42).

M10. Graduate courses. The PathChooser fellows are expected to follow the PhD/specialisation graduate courses, as required by their host institution/university, or at any of the other PathChooser institutions. D5.33: Report of fellows completion of graduate courses (Mo 28).

M11. Participation of fellows to training courses and activities. The fellows will participate to training courses, including compulsory courses of complementary skills, and courses on specialised techniques or instrumentations. In addition, they will be encouraged to participate to training opportunities outside of PathChooser, under the guidance of their supervisors (job fairs, industry exhibitions, lectures courses etc.). D5.34-36: Periodical reports of fellows participation to training courses and activities (Mo 15, 24, 33).

M12. Mid-term and final summing up. Mid-term and final summing up of the training course (critical evaluation of training activities, fellows’ outcome, career development and implementation planning). D5.37-38: Mid-term and final reports of PathChooser training outcomes (Mo 24, 48).

Types of training offered:

The training structure will comprise local or in-house and network-wide activities as well as secondment. Locally, fellows will attend scientific lectures as well as existing courses in oral presentation, scientific writing, patent issues, and language classes. Considerable provisions have been taken so that they will be included in local teaching programmes, e.g. ongoing (English language), PhD courses in specified areas of nanomedicine, bionanointeractions, molecular imaging

Creating networks between specialists from different areas through joint initiatives is the basis to defeat fragmentation of knowledge. We will therefore organise two open conferences after 1.5 and 3 years of the programme. The first conference, tentative title “In vitro-in vivo correlation in models of biological barriers”, will be held (location to be decided) and will address the need to ensure that in vitro models capture the essence of in vivo barriers, and transcytosis. The second conference, tentatively titled “Designing the bio-nano-interface for drug-delivery carriers” will be held in Cyprus. At least 20 participants from outside the consortium, either as invited speakers or as attendees, including representatives of the pharmaceutical industry, biotechnology companies, and relevant regulatory agencies..

Partner 5 (EPOS) will organize at one of the later 6-monthly meetings (E.g. Mo30) a Meet-the-Manager Workshop, where fellows will present their findings to company managers. This will be an important opportunity to train fellow on communication with non-scientific audiences and to facilitate inter-sectoral interaction and networking. The event will also include lectures from senior industrial staff on Industrial perspectives on nanotechnology and on Structuring research careers in industry. Participants will include senior staff from the pharmaceutical industry which will cover the topic From Bench to Bedside development, and will also include a session on Planning and implementing clinical trials. ESRs/ERs will also benefit from their participation in the local Entrepreneurship Competition enabling teaching modules (series of lectures and workshops on business plan writing and presentation), facilitated by EPOS and the DIOGENES incubator, UCY through the Cyprus Entrepreneurship Competition that takes place annually (Section B5 describes the outreach planned).

Each of the partners is renowned experts in their arenas and all have impressive records of advisor of researchers. In all cases, PathChooser ESR and ER Fellows will have a nominated second scientific

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advisor, at one of the other partner institutes, to cover additional expertise areas required by the fellow to complete their individual training plan. Finalisation of these supervisors will be made in conjunction with the completion of the Fellow’s Individual Career Development Plan2.

Each of the partners is renowned experts in their arenas and all have impressive records of supervision of researchers. In all cases, PathChooser ESR and ER Fellows will have a nominated second supervisor, at one of the other partner institutes, to cover additional expertise areas required by the fellow to complete their individual training plan. Finalisation of these supervisors will be made in conjunction with the completion of the Fellow’s Individual Career Development Plan.

Personal career development. At the start of their contract, fellows will receive a Manual for Fellows, prepared by the responsible MC members (see B.4.3). The Manual will contain information about the FP7-PEOPLE-2012-ITN programme, the PathChooser project structure, timeline, procedures and communication routes, and about good scientific practice including GLP and ethical standards. The Manual will also inform about scientific writing, job perspectives (including reference to the Researchers Mobility Portal for the European Commission (http://ec.europa.eu/euraxess), administrative questions like taxes, social security and pensions, and gender issues.

A Personal Training Plan will be set up individually for each fellow, that will include the scientific project and the tutor assigned (including a second tutor when required by the relevant PhD programmes), the timeline of training, the secondments to other institutions, contacts with industrial partners, and a list of skills to be obtained during the Fellow’s training. Checkpoints will be both scientific (M3: Fellows’ seminars) and related to training (M9: Participation to journal clubs and lectures). A quality assessment questionnaire will be given to the fellows at the end of all meetings and training events in order to assure that training is tailored to the fellows needs. The questionnaire should probably be a deliverable.

All ERs and ESRs will receive approximately of 4 weeks of training in complementary skills which will be in addition to the specialised research methodology transfer that will be part of their spending time between partner institution as appropriate to their individual research projects. Research projects have been tailored so that all ESR/ERs would be expected to spend a significant period (e.g. 6/4 months) in a partner laboratory in another country acquiring specialized skills which can then be transferred (transfer of technology/know how) to the host laboratory i.e. primary laboratory where the researcher is based. These details are described within the research sub-projects for the individual fellows.

A dedicated Supervisory Board (SB) consisting of all MC members plus outstanding representatives from academic institutions, industry and regulatory agencies will provide external input and monitor the progress of the training and research programs, as described in detail in Section B5. This body will be responsible to oversee the progress of the PhD training for the ESRs and the career development of the ER, and to ensure that the quality of the training meets international standards of best practice. There will also be a representative of the ESRs on this Board, to ensure that the ESR/ER play an active role in decision making, training planning etc.

The List of secondments planned is given in Section B4, Table B.3.1.2 which highlights also what each fellow will gain for their own personal training and development plan. A summary of this host-specific training, which can be accessed by the fellows via their secondments, is given in the Table below:

2 Some institutions do not accept formal supervision of students from other institutions, so this system of a secondary advisor is maintained within the network

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Partner Host-specific skills in which training will be provided to hired fellows / secondees NUID UCD NUID UCD have developed novel concepts around the role of the bionanointerface and its role in

determining nanoparticle fate and behaviour in living systems. Methods to characterise nanoparticle-protein and nanoparticle-cell interactions in quantitative manners will be shared by NUID UCD.

SDU SDU will provide training in the use of naturally occurring human pattern recognition molecules for the development of nano-carriers, muco-adhesive and cell-entering targeting devices. This will complement the otraining in other targeting modalities provided by e.g. NUID UCD, UNIMA etc.

HZI Expertise and research at HZI focusses on the important issue of cell an tissue based in vitromodels, and the optimisation of such models in order to predict the in vivo behaviour. Researchers will be trained in these key issues, and in approaches to correlate responses from healthy versus diseases barriers to nanocarriers.

KCL THE KCL team have years of expertise in assessing the inteactions of nanoparticles with the Blood-Brian Barrier in vivo and the development of realistic in vitro models, and will provide training training in glioblastoma spheres isolation in primary cultures and in in vitro models of transcytosis.

EPOS Industry exposure and research in an industrial contextEPOS have unique expertise in models that include a mucus component, which is often lacking in culture models. Training in mathematical modelling of nanoparticle transport can also be shared.

MPI-CBG MPI-CBG are focussed on mechanisms of endocytosis and will provide training in: 1) the mechanisms underlying endosome biogenesis, 2) how endocytic transport regulates and is modulated by intracellular signalling and 3) the regulation of endocytosis in polarised cells, such as epithelial barriers.

UNIVBRIS UNIVBRIS will provide training in specific features of the foetal barrier model, to enable detailed imaging of uptake and translocation, and comparison with air-lung and blood-brain barriers.

UNIMA Fellows hosted or seconded at UNIMA will learn a wide variety of therapeutic and diagnostic modalities, including development of Apo-lipoprotein functionalised nanocarriers and the role of geometry and orientation aspects in the optimisation of the targeting and transport efficiency.

ACS Industry exposure and research in an industrial context; The ACS training contribution shall be the development of advanced cell culture models of human tissues, which shall provide the ACS fellow with training in cell biology and commercially-orientated cell culture and analytical technologies.

Table 3.1.1 Recruitment Deliverables per Participant

Participant NoEarly-Stage Researchers Experienced Researchers

Number of Researchers

Total Researcher Months

Number of Researchers

Total Researcher Months

1 2 72 0 02 1 36 0 03 1 36 0 04 1 36 0 05 1 36 1 246 1 36 0 07 1 36 0 08 1 36 0 09 0 0 1 24(Sub-) Total 9 324 2 48Meaningful exposure of each researcher to another sector, in particular through secondments

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Table 3.1.2 – List of SecondmentsList of Secondments

Fellow No.

Host Beneficiary

Place of Secondment

Purpose

ESR1 NUID UCD MPI-CBG

ACS

Training in advanced microscopy and imaging methods coupled with biochemistry of endocytotic processes.Industry exposure; Development of cellular assays for assessing uptake.

ESR2 SDU NUID UCD

HZI

EPOS

Training in biointerfacial aspects and understanding non-specific binding and protein corona.Industry exposure; Assessment of the effectiveness of targeting strategy in vitro/in vivo.Training in the assessment of the effectiveness of targeting strategy in vitro/in vivo.

ESR3 HZI EPOS Industry exposure; Cross-validation of lung, gut and brain barrier model responses to test carrier; model validation for mucus barrier.Training in biointerfacial aspects and understanding non-specific binding and protein corona.

ESR4 KCL UNIMA

NUID UCD

EPOS

Development of Apo-lipoprotein functionalised nanocarriers –geometry and orientation aspects.Training in biointerfacial aspects and understanding non-specific binding and protein corona.Industry exposure; Assessment of the effectiveness of targeting strategy in vitro/in vivo.

ESR5 EPOS NUID UCD

MPI-CBG

Academic exposure: Correlation of responses of healthy and diseases brain barriers to nanocarriers.Academic exposure: Training in synthesis and functionalisation of nanocarriers for targeting.

ESR6 MPI-CBG UNIVBRIS

ACS

Training in specific features of the foetal barrier model, to enable detailed imaging of uptake and translocation.Industry exposure; Correlation of uptake mechanisms and organ/tissue dose in vitro/in vivo.

ESR7 UNIVBRIS EPOS

NUID UCD

Industry exposure; Assessment of the effectiveness of targeting strategy in vitro/in vivo.Training in biointerfacial aspects and understanding role of protein corona in uptake/barrier crossing.

ESR8 UNIMA HZI

ACS

Complex epithelial cell model and assessment of carrier interactions and uptake.Industry exposure; Development of cellular assays for assessing uptake.

ESR9 NUID UCD UNIMA

SDU

Training in design and surface modification of nanocarriers for interaction with biological barriers.Training in design of biomimetic targeting moieties for nanoparticle targeting. (Industry secondment to Sanofi)

ER1 ACS NUID UCD

EPOS

Academic exposure: Training in biointerfacial aspects and role of protein corona in uptake/transcytosis. Academic exposure: Development of cellular assays for assessing uptake.

ER2 EPOS KCL

SDU

Academic exposure: training in glioblastoma spheres isolation in primary cultures and in in vitro models of transcytosisAcademic exposure: Training in advanced microscopy and imaging methods for endocytosis.

B.3.2. Network-wide training events, schools, conferences, workshops

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Meetings and training events will be organised by partners, to ensure that the consortium and all fellows meet to discuss their research at least 6 monthly. The meetings with the ESR / ER will also include elements of training in transferrable skills, with a preliminary schedule given below.

Table 3.2.1 - Training activities

Main Training Events & ConferencesLead

Organising Institution

Planned date

Planned venue

1Kick-off meeting (MC, SB)• Plan on advertisement of fellows’ vacancies• Agreement on the fellows’ selection procedures• Implementation of management structure• General discussion on administration and finance

NUID UCD 2Cyprus

2Fellows kick-off meeting, MC meeting (MC, tutors, and fellows)• Fellows and tutors meeting and introduction • Illustration of the general project• Illustration of the specific projects• PhD Induction Program and Knowledge Protection• PhD Training on the Bio-nano interface & multi-disciplinary research skills and approaches• Second meeting of the MC

SDU (& NUID

UCD)

7Dublin

3Progress meetings (MC, SB, tutors, and fellows)• Progress of the project • Fellows’ scientific presentations • Main problems encountered, go/no go decisions• Assessment of need for reorientation of training• Transferrable skills training 1 (M12): Scientific writing & Communication with different audiences• Transferrable skills Training 2 (M36): Project management & Grant writing skills

MPI-CBG

UNIVBRIS

12

36

Germany

United Kingdom

4International Conference + Progress meeting (MC, SB, tutors, fellows, and invited scientists)• International conference on Mechanisms of nanoparticle passage

across biological barriers• Progress meeting as above• Outreach activities from fellows

KCL 18 London

5 Complementary skills training courses (local hosts, and fellows)Challenges in a Commercial Environment • IPR protection, legal matters, Open Innovation• Product development and validation• Planning and implementing clinical trials• Compliance and regulatory aspectsNano-Biotechnology Entrepreneurship• Writing a business plan• Sources of finance & pitching to potential investors• Establishment of a virtual spin-out company by fellows etc.

ACS

EPOS

18

30

Dublin

Cyprus

6 International Conference + Fellows conclusion meeting (MC, SB, fellows and invited scientists)• International conference on Therapeutic strategies involving

nanoparticles actively transporting across biological barriers• Presentation of final training results • Fellows’ final scientific presentations

HZI 36 Germany

7 Final workshop (MC and SB)• Conclusions (will be included in the final report) • The future

NUID UCD 42 Dublin

The training structure will comprise local or in-house and network-wide activities as well as secondment. Locally, fellows will attend scientific lectures as well as existing courses in oral presentation, scientific writing, patent issues, and language classes. Considerable provisions have been taken so that they will be included in local teaching programmes, e.g. ongoing (English language),

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PhD courses in specified areas of nanomedicine, bionanointeractions, molecular imaging, and various institutions offer relevant specialized courses.

Complementary skills training courses across both sectors (academia and SMEs) will be evaluated and it is expected in the future to have delivery across institutions as partnerships develop either as distance learning options to prevent duplication of courses or for collaborators to extend in the future (beyond the time frame of this proposal) the delivery of these courses in partner host institutions thus developing lasting academic and industrial collaboration for delivery of complementary skills courses, embedded in a PhD Program in Nano-BioInteractions and Drug delivery across biological barriers (B5). Intended examples include;Course 1. PhD Induction Program and Knowledge Protection (SDU and NUID UCD, 5 days, month 7)

Course 2. Challenges in a Commercial Environment (ACS, NUID UCD and Genzyme, month 18)

Course 3. Nano-Biotechnolnogy Entrepreneurship (EPOS, in conjunction with the University of Cyprus-based Associate Partner, business incubator “Diogenes”, 5 days: month 36)

B.4 Implementation

B.4.1. Workplan

Overview of the WorkplanThe PathChooser project is divided into 4 research and 1 Training WP. Model nanoscale carriers will be designed, prepared, labelled and functionalised with exemplar therapeutics (WP3) and assessed in WP1 and WP2 for their interactions (uptake, export - if occurring - and transcytosis) in vivo and using model biological barriers and cellular barriers, with inter-laboratory validation of key results. Materials and biologicals will also be checked and distributed centrally (by partner 1) thereby ensuring reproducible results.

WP1 - Novel Nanocarriers Design, Development, Synthesis. Leader: SDU Deputy: UNIMA

Development of novel nanocarriers for barrier crossing; A set of model nanocarriers (polymeric, silica etc.), monodisperse, controlled size (in the range 20-250 nm), shape (spherical, prolate, oblate, long axial ratios), reference surface targeting grafted ligands (e.g. for brain, Apolipoprotein E) will be prepared (in an iterative manner by assessment against the various biological and cellular barriers) in order to identify key transport moieties / patterns and pathways. Internal ‘dot’ labels allowing for fluorescence, NIR, isotope, magnetic, MRI, PET and other imaging approaches will be incorporated to allow time- and space-resolved tracking. These particles to be checked for quality etc. centrally, and all dispersion protocols etc. will be applied identically (where feasible) by all partners to ensure cross-comparability of the outputs in WPs 2, 3.

WP 2 - Mechanisms of uptake of biomacromolecules across cellular and non-cellular barriers . Leader: HZI Deputy: MPI-CBG

Clarification of in vitro endo- and exocytosis processes, and cross platform (all Partners) validation; Key in vitro, cell-level models, including the environmental factors (surfactant, mucus, mucosa, extracellular matrix etc.) cross-correlated using high quality model materials Organ- / cell-level (using imaging, tissue disassembly, cell separation etc.) biokinetics of several nanocarrier sizes, shapes. Correlation of uptake/tissue localisation with cell type and bionanointerface.

WP 3- Mechanisms of uptake of biomacromolecules across biological tissue barriers (in vivo, multi-cell and other models). Leader: KCL / Deputy: UNIVBRIS

Clarification of in vivo endo-, exo- and transcytosis processes, and cross platform (all Partners) validation; Key in vivo, cell-level and other models to be cross-correlated using high quality model

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materials, varying key parameters of program. Reference States in vivo; Identification and classification of several nanocarrier transport processes across blood brain, air lung, and gut barrier models (in some cases-carried out independently at two sites, e.g. for BBB: porcine and mouse (normal and diseased) brain perfusion, intravenous infusion, specific knock-outs, correlation of uptake/tissue localisation with structure and bionanointerface.

WP4 - Route, organ and tissue specific delivery strategies. Leader: NUID UCD Deputy: MPI-CBG

Platform for data storage, integration and interpretation; This workpackage will ensure that the data generated by the different partners can be captured in an appropriate manner (using living templates), to ensure its suitability for modelling, for cross-barrier and cross-mechanisms of action comparisons, and to enable pattern recognition and clustering of responses based on nanocarrier physico-chemical or biointerfacial properties.

WP 5 – Implementation of Fellows Training Leader: EPOS Deputy: NUID UCD

Ensuring quality control across all aspects of the pathfinder research, training and reporting; This workpackage is designed based on the experience of (with support from) the QNano EU Infrastructure, coordinated by Dawson, and covers both management of fellows training, project management and materials and data management, due to the unique considerations of nanocarriers in terms of quality control issues.

Private sector involvement at the highest possible level appropriate to the research topic

As described in Sections B2 and B3, all fellows will be given multiple opportunities to engage in cross-sectoral training, with fellows appointed at academic institutes undertaking a period of secondment at one of the industrial partners, and vice versa.

Additionally, industry partners will be central to the training elements. Thus, throughout the training schools and workshops, a significant element of the teaching and training will be delivered by companies. This will include technical issues of the science, but seen through a perspective of industry partners, and the different drivers (e.g. corst, regulatory hurdles) they face, rather than purely technical needs.

This involvement will extend also to IP management, development of entrepreneurial skills, understanding of generic commercial constraints and the potential for open innovations etc.

Some examples of transferrable skills training courses, are described in Section B3, such as Course 2. Challenges in a Commercial Environment (ACS, SME and Genzyme, month 18) and Course 3. Nano-Biotechnolnogy Entrepreneurship (EPOS, in conjunction with the University of Cyprus-based Associate Partner, business incubator “Diogenes”, 5 days: month 36)

How essential is non-ICPC Third Country participation, if any, to the objectives of the research training programme

Given the global nature of research, medicine and regulation, and the desire of the consortium to provide maximum opportunities for the ESR and ER Fellows in terms of their career development, the consortium have chosen to include a US-based company (Genzyme, part of the global company Sanofi which has extensive research activities in France and Germany, as a level 2 partner, who will support in the provision of transferrable skills training, and will provide a global, and large industry perspective for the fellows. Genzyme has role in the therapeutic application of this science, and will provide a helpful technical linkage with Sanofi, especially with the CNS research group based in France. We will investigate ESR/ER secondment opportunities there.

Clarity of the plan for organising training events (e.g. workshops, conferences, training courses).

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Table 3.2.1 summarises the main events planned for the ESR/ER Fellows and their timetable. The main aspect of note is that the fellows and the entire project team will meet every 6-months, which will both promote networking and relationship-building between the fellows and the senior scientists, and also provide a mechanism to continuously review the research and training progress of the ESR/ER Fellows.

As described in detail in section B5, each ESR/ER Fellow will have responsibility to develop (in agreement with their supervisors, and with approval from the Supervisory Board) and individual Career Development Plan, which will tailor their training plan to their individual career goals and aspirations, and to their background skills and knowledge, ensuring the development of complementary skills and knowledge during the ITN training.

The various workshops and training events provide a focus to monitor and review all of these activities, but they can also provide an excellent opportunity for the training of the fellows. Thus, it will be usual in each of the events that the fellows themselves assist in the organization of all aspects. At least one such event will be entirely organized by the fellows themselves, along the lines and style of a 'Gordon' or ESF research conferences. That is, fellows will elect those primarily responsible for different functions (based on a 'bid' by different interested fellows made early in the program) and thereafter, all aspects will devolve to them, and the general guidance of the fellows. As much as possible PI's will merely provide advice when required, and all details (including aspects of financing-though under overall guidance of the Manager) will be handled by fellows.

Table 4.1 - Project titleResearcher No.

Project Title Host Institution

Relevant Work Package(s)

Duration (months)

Indicative start date

ESR1 From endocytosis to transcytosis: investigation of key steps and signals

NUID UCD WP2 / WP3 36 M2

ESR2 Harnessing Natural Pattern Recognition Mechanisms for Macromolecular Drug Delivery

SDU WP1 / WP2 36 M6

ESR3 Interaction of nanoparticles with the pulmonary barrier; the role of mucus in health and disease

HZI WP2 / WP3 36 M7

ESR4 Apolipoprotein E and A targeted albumin nanoparticle delivery to the CNS: Mechanism and route of delivery

KCL WP3 36 M7

ESR5 Optimizing the transport of nanoparticles through diseased mucus

EPOS WP4 36 M7

ESR6 Development of bio-inspired routes for delivery of siRNA

MPI-CBG WP2/WP3 36 M7

ESR7 Nanoparticle exposure of placental barriers. An indirect route of teratogenesis

UNIVBRIS WP3 36 M1

ESR8 Design of degradable targetednanoparticle-based delivery vehicles

UNIMA WP1 36 M1

ESR9 Designing the bionanointerface for transcytosis and barrier crossing

NUID UCD WP1 36 M2

ER1 Cell-Based Nanosafety Assay Design and Validation for Accreditation and Dissemination

ACS WP3 24 M7

ER 2 Experimental testing of nanoparticulate systems for crossing intact and disrupted barriers

EPOS WP3 20 M12

The network as a whole undertakes to provide a minimum of 372 person-months of Early Stage and Experienced Researchers whose appointment will be financed by the contract. Quantitative progress

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on this, with reference to the table contained in Part C and in conformance with relevant contractual provisions, will be regularly monitored at the consortium level.

B.4.2. Fellows individual research projects

If necessary and upon approval by REA, the experts listed in this Description of Work might be replaced with scientists with equivalent expertise

FellowESR1

Host institution NUID UCD

Duration36 months

Start dateM2

Project title: From endocytosis to transcytosis: investigation of key steps and signals; WP1, WP3Supervisor name: Kenneth Dawson (NUID UCD); Co-supervisor: Marino Zerial (MPI-CBG)PhD enrolment: Y

Objectives:This ESR 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 biological barrier in question, and a second will be utilized to drive the nanoparticle out of the endolysosomal pathway andtowards the transcytotic pathway (e.g. ApoE or transferrin). For example, it has been shown that brain capillary ECs bind and internalize but do not degrade LDL. Specific objectives are:O1: To develop a standardized platform determining endocytosis/transcytosis efficacy for nanomaterialsO2: To identify targeting strategies that works in vitro

Tasks and methodology: T1: Development of a standardized platform determining endocytosis/transcytosis efficacy for nanomaterialsT2: Identification of the most promising intracellular targeting moieties, via a focused screen on peptides or chemical scaffolds for transcytosis using an in vitro system (e.g. polarized primary hepatocytes, liver and brain sections) and validating the most promising targeting moieties in vivo.T3: To test the trafficking properties of the devices developed in the other projects, and to forward devices for testing in specific model systems e.g. to P2-4, 7, and 9-11.The main methodologies to be utilized by the ESR are molecular and cell-based assays, and their application to investigating of mechanisms of nanomaterials endocytosis and transcytosis.

Results: R1: Identification of targeting moieties that have the potential to workR2: Identification of a targeted carrier that works in vitroR3: Ph D ThesisDissemination:

1. Participation in three international conferences during M22 – M36.2. At least one research article to be published from M29 to M36.

Planned secondment:S1: MPI-CBG: Training in advanced microscopy and imaging methods coupled with biochemistry of endocytotic processes. 3 months in Year 2S2: ACS: Industry exposure; Development of cellular assays for assessing uptake. 3 months in Year 3Risk assessment:This project is partly dependent on the success of carriers developed by other projects. Should such carriers not be successful, this project will focus on identifying the detailed mechanism for their unsuccess.

FellowESR2

Host institution University of Southern Denmark

Duration36 months

Start dateM6

Project title: Harnessing Natural Pattern Recognition Mechanisms for Macromolecular Drug Delivery; WP2, WP3Supervisor name: Prof. Jan Mollenhauer (main supervisor), University of Southern Denmark (SDU), Prof. Kenneth Dawson (co-supervisor), University College Dublin (NUID UCD)PhD enrolment: Y

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Objectives:O1: Testing of pattern recognition (PR) peptide and protein interactions with nucleic acids (month 15)O2: Analysis of PR peptide and protein interactions with receptors on cancer cells/cancer stem cells (month 24)O3: Optimization of PR peptide internalization (month 36)O4: Testing of PR peptide and protein-mediated siRNA delivery in vitro (month 42)

Tasks and methodology: T1: Development of an ELISA-based detection assay for PR peptide/protein interactionsT2: Development of sensitive assays for PR peptide/protein internalization into cancer cells/cancer stem cells

Results:R1: ELISA-based detection assay for PR peptide/protein interactions with ligandsR2: Assay for PR peptide/protein internalization into cancer cells/cancer stem cellsR3: PhD thesis

Dissemination: Publications prospectively will be filed on the interactions studies of PR peptides/proteins with siRNAs/cells as well as on PR peptides optimized for siRNA delivery (in the case of success). It is planned to file a patent on the latter. Participation in two large international conferences in the field of nanomedicine is planned with poster presentations of the data.

Planned secondment:S1: NUID UCD, month 16-18 (3 months) for studying PR peptide/protein interaction with serum componentsS2: HZI, month 33-35 (3 months) for analysing PR peptide/protein internalization into normal epithelial cells/at mucosal surfacesS3: EPOS, month 27 (0.5 months) for assessment of the effectiveness of targeting strategy in vitro/in vivo.Risk assessment: No particular risks identified at the present time point.

FellowESR3

Host institutionHelmholtz Centre for Infection Research (HZI) / Helmholtz-Institute for Pharmaceutical Research

Saarland(HIPS)

Duration36 months

Start dateM7

Project title: Interaction of nanoparticles with the pulmonary barrier; the role of mucus in health and disease (WP 2 and 4)Supervisor name: Claus-Michael Lehr (HZI) and Andreani Odysseos/Triantfyllos Stylianopoulos (EPOS)PhD enrolment: Y, Saarland University, Saarbrücken, Germany.

Objectives: 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,etc.,that allows: O1: To deposit aerosolized nanoparticles (e.g. to be developed by the project partners) at an air-interface;O2: To study the role of mucus and surfactant as non-cellular elements of the pulmonary barrier;O3: To address pathological changes in such model and to nanoparticle transport under the influence of some diseases such as inflammation and/or bacterial infection.

Tasks and methodology: T1: Set up a cell culture model of the airway epithelium that develops an adherent mucus gel layer; use Calu-3 cell line at starting point, explore co-culture option with mucus secreting and ciliated cells.T2: Quantify mucus productions, thickness and clearance of the gel layer under different physiological conditions (e.g. air-liquid versus liquid-liquid interface) including pathological changes such as, inflammation and/or infection. T3: Explore and validate the transfer of the cell culture into air-aerosol deposition chamber (e.g. PADDOCC, ALICE, etc.).T4: Study the deposition, transport and clearance of the nanoparticles in mucus, compared with binding andpossible uptake by epithelial cells and macrophages. T5: Address the possible role of physiological and pathological changes (e.g. inflammation, infection, etc.) on the aforementioned processes, including also the role of pulmonary surfactant and its proteins.

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Results: R1: A cell culture model of the upper respiratory tract suitable for depositing aerosolized particles under controlled conditions at an air-interface.R2: A method to detect and quantify particle movement through the bronchial mucus gel layer and subsequent interaction with epithelial cells.R3: A better understanding of physiological and pathological factors governing nanoparticles transport through bronchial mucus and eventually across the air-blood barrier.

Dissemination: The results will be presented at international scientific meetings and submitted to international peer reviewed journals. Patent protection will be sought first to secure generated intellectual property rights. Besides regular project reports the student will write a PhD thesis and submit it to Saarland University.

Planned secondment: The student will spend some 3 months at the partner EPOS, the precise date is still to be determined in interaction with EPOS and based on the progress of the project of ESR5. Artificial mucus will be developed in combination with the cell culture model and the algorithms developed by ESR5 will be validated in our model.

Risk assessment:The necessary equipment and supplies are either available at HZI or will be purchased from the project budget. The planned cell culture model bears appreciable technical risks; in case it cannot be realized, artificial mucus maybe added or cell free mucus preparations will be explored as alternatives.

FellowESR4

Host institution Kings College London

Duration36 months

Start dateM7

Project title: Apolipoprotein E and A targeted albumin nanoparticle delivery to the CNS: Mechanism and route of delivery; WP3, plus WP1 and WP4Supervisor name: Dr David J Begley, Dr Sarah Thomas, Kings College LondonPhD enrolment: Y

Objectives:Recently we published two papers (Zensi et al. (2010) J Drug Target 18: 842-848, Zensi et al. (2009) 137: 78-86) demonstrating that 200 nm albumin nanoparticles could be delivered across the blood-brain barrier (BBB) in both rats and mice by targeting with either ApoE or ApoA1 and that these nanoparticles were delivered to the cytoplasm of neurones. These experiments have established a number of important principles. For the use of these particles for drug delivery to the CNS, however, a number of questions still remain unanswered:O1: It is not clear how these nanoparticles reach the cytoplasm of the neurones. The particles probably do not travel in the extracellular space of the brain as the width of the intercellular space is on average 70 nm and the particles are too large to move freely in this space. We can observe by transmission electron microscopy the particles within the endothelial cells forming the blood-brain barrier and within the extracellular matriximmediately surrounding the capillaries. In brain parenchyma we have not convincingly observed particles in the extracellular cleft and all particles seen appear intracellular.O2: The question then arises: is further transport of the nanoparticles in the brain via an intracellular route? The astroglia seem well positioned to fulfill this role as their end feet completely surround the blood-brain barrier capillaries and their further processes abut neurones to which the particles could be delivered. It has recently been suggested that the astroglia may form pathways in the brain of this nature (Begley DJ. (2012) Brain Superhighways. Sci Trans Med 4(147): 147fs29. doi:10.1126/scitransmed.300461). This hypothesis will be tested with the albumin nanoparticles using carefully timed electron microscopy after intravenous administration.O3: A further question is: do the albumin nanoparticles present in the cytoplasm of neurones still have their apolipoprotein target attached or is this removed or modified during transcytosis?O4: We will also determine the ability of these targeted nanoparticles to deliver radiolabelled drugs to specific brain regions.O5: These studies will provide important information on the mechanism and route of delivery of albumin nanoparticles to specific regions of the CNS and evaluate their utility as a drug delivery system for treating many disorders of the CNS where the blood-brain barrier presents a formidable obstacle to successful treatment.

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Tasks and methodology:T1: The student working on this project will further investigate the routing of the apolipoprotein targeted albumin nanoparticles through the BBB and CNS using a combination of in vivo and in vitro techniques developed at KCL and employed to study transport across the blood-brain barrier.T2: This will enable us to determine the time course and route of delivery of the albumin nanoparticles to neurons and the mechanism of transcytosis across the blood-brain barrier.T3: We will then apply these techniques to the transport vectors in use at NUID UCD, UNIMA and EPOS.T4: The ability of these delivery systems to deliver radiolabelled drug to the CNS will be evaluated in both in vivo and in vitro studies and the efficiency of the different delivery systems compared head-to-head.

Results: Year 1: Elucidation of the route and mechanism of apolipoprotein-targeted albumin delivery to neurones of the CNS.Year 2: Comparison of albumin nanoparticle delivery to the nano- systems of NUID UCD, UNIMA and EPOS.Year 3: Comparison of the effectiveness of the available nano-systems to deliver radiolabelled drug to the CNS

Dissemination: 1. The results of the study will be disseminated via peer-reviewed research journals, international

meetings, network meetings and public domains. 2. Patents will be applied for when and if appropriate.3. At least two peer-reviewed research papers will be published during year three.

An international conference will be organised at Kings College M24 (Table B3.2) at which results will be presented and the Fellow will be xpected to attend and participate in at least one international meeting during each year of their study.

Planned secondment:UNIMA, NUID UCD and EPOS. The student will spend approximately a month at each institution during the second year of study to learn the fabrication techniques of the nano-systems developed by these groups so that the methodology described above can be applied to these constructs to determine commonality and differences in the mechanisms and routes of delivery to the brain and their comparative ability to deliver drugs to specific brain regions.Risk assessment:There would appear to be little risk attached to this study as the resources and expertise required are currently available at Kings College London and expertise with the other nano-systems is possessed by the partners.

FellowESR5

Host institution EPOS-Iasis, R@D

Duration36 Months

Start dateM7

Project title: Optimizing the transport of nanoparticles through mucous biological tissues (WP4 and WP1)Supervisor name: Andreani Odysseos/Triantafyllos Stylianopoulos (EPOS) and Kenneth Dawson (NUID UCD)PhD enrolment: Y- PhD degree from University College Dublin Objectives: O1: Development of a mathematical model for transport of NPs through the mucus O2: Validation of model predictions with in vitro experimental dataO3: Development of design rules for optimal penetration of nanoparticles through diseased mucus layer (e.g. cystic fibrosis).

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Tasks and Methodology: T1: Develop stochastic models to predict the diffusivity of nanoparticles based on the micro-structural characteristics of the mucus (viscosity, fiber diameter and volume fraction) and the physical properties of the particles (size, surface charge or shape), taking whenever possible into account steric, hydrodynamic and electrostatic interactions between the nanoparticles and the fibers: Representative volume elements (RVEs) will be constructed in silico to provide a statistical representation of the mucus microstructure. A random walk approach will be employed to simulate the diffusive transport of nanoparticles of varying size, surface charge or shape in these RVEs. A Stokesian dynamics method will be used whenever possible to account for steric and hydrodynamic interactions between the particles and the mucin fibers. Electrostatic repulsion will be also considered for the diffusion of spherical particles within a periodic array of perfectly aligned fibers.T2: Validate model prediction with in vitro experiments in artificial native and diseased mucus. Model predictions will be validated with in vitro experiments in artificial mucus both for the native and diseased case. Fluorescently labeled nanoparticles of different size, surface charge, or shape will be employed provided by ESR8 in WP1 and measure their diffusion coefficient using fluorescence microscopy techniques and particle tracking analysis. T3: Use model predictions to derive design rules for the properties of nanoparticles (i.e., size, shape and charge) that optimize penetration through the mucus layer: Simulations will be run to predict optimal nanoparticle properties that ensure effective penetration in the mucus layer. Results from the stochastic models will be communicated to partners UCL, project ESR8, as well as partner HZI, project ESR3, in order to correlate data from the mucus barrier with data from the air/blood barrier. Results -Deliverables:R1: PhD thesis R2: Report on mathematical modeling of diffusive transport in mucus R3: Report on design rules for optimal penetration of nanoparticles to mucus

Dissemination: 1. Participation in three international conferences during M30 - M42.2. At least one research article to be published from M36 to M42.

Planned secondment: ESR5 will have two, three-month secondments at NUID UCD and MPI-CBG. At NUID UCD ESR5 will expand the experimental validation protocol for assessment of nano-biointeraction in respiratory and intestinal epithelial cells in presence and absence of mucous. At MPI-CBD ESR5 will complement the mathematical modelling with data from intracellular trafficking under different conditions, in order to provide predictions of diverse perturbation.

Risk assessment: Particle tracking analysis for diffusion measurements of nanoparticle formulations is a new and promising technique but faces certain limitations. If this method will not work properly for our application, we will measure diffusivity using fluorescence microscopy. There is not yet an established method to account explicitly for hydrodynamic and electrostatic interactions of non-spherical particles. In our analysis, we will extend existing methodologies to incorporate these parameters. Alternatively, we will account explicitly only for steric interactions and implicitly for hydrodynamic interactions.

FellowESR6

Host institution Max Planck Institute of Molecular Cell Biology and

Genetics (MPI)

Duration36 months

Start dateM7

Project title: Development of bio-inspired routes for delivery of siRNAs (WP2/WP3)

Supervisor name: Marino Zerial (MPI-CBG) and Colin Wilde (ACS)

PhD enrolment: Yes: 'IMPRS-CellDevoSys, TU Dresden awards degree'Objectives:O1: Develop novel bio-inspired strategies for the delivery of siRNAs into cells for therapeutic applicationsO2: Optimize siRNA cellular targeting; O3: Optimize siRNA endosomal escapeO4: Optimize siRNA sub-cellular targeting.

Deleted:

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Tasks and methodology:The ESR fellow will follow a pipeline already established in the Zerial lab, using the mouse liver as a model system. (S)he will:T1: Visualize the intracellular fate of siRNA formulations in vitro and in vivoT2: Monitor siRNA escape from endosomesT3: Screen chemical compounds and RNAi libraries in high content cell-based image analysis assaysT4:Apply image analysis and statistics to validate the candidate formulations. Results: R1: Strategies for optimized cellular targeting of siRNA formulationsR2: Strategies for optimized endosomal escape of siRNA formulationsR 3: Strategies for optimized sub-cellular targeting of siRNA formulationsR4: PhD ThesisDissemination:One report at M36 to M42Planned secondment: UNIVBRIS: Training in specific features of the fetal barrier model, to enable detailed imaging of uptake and translocation. 3 months in Year 2ACS: Industry exposure; Correlation of uptake mechanisms and organ/tissue dose in vitro/in vivo. 3 months in Year 2Risk assessment: The main challenge of this project is to design novel siRNA technology with high delivery efficiency. The risk is that the formulations that will be tested will not lead to a substantial increase of efficacy of siRNA delivery over the current systems. However, we have ideas on how to improve the current technology, either through lipid nanoparticles or chemical conjugates. If those ideas prove wrong, we will use current formulations to characterize their intracellular trafficking.

FellowESR7

Host institution Bristol University

Duration36 Months

Start dateM1

Project title: Nanoparticle exposure of placental barriers. An indirect route of teratogenesis (WP3 and WP4)Supervisor name: C. Patrick Case (UNIVBRIS) and Kenneth Dawson (NUID UCD)PhD enrolment: Y- PhD degree from University of BristolObjectives: O1: Improve the cell barrier to make it a more faithful replica of the actual placentaO2: Explore the mechanism and actions of the signallingO3: Use model predictions to derive design rules for the properties of nanoparticles (i.e., size, shape and charge) that optimize signaling across the barrier:Tasks and Methodology: T1: Improve the cell barrier to make it a more faithful replica of the actual placenta.To do this we will isolate primary trophoblasts from human placenta and use them to makebilayered and monolayered barriers with and without syncytialisation. Up until now this has not been possible due to the lack of cell division of these primary cells. However using novel technology we have recently succeeded in creating primary trophoblast confluent cell barriers on transwell inserts and wish to extend this work.We have good access human placenta in our hospital based laboratory and will therefore be able to compare the structure and the signalling of these model cell barriers with the real placenta.We will validate the barriers with confocal imaging of e cadherin and ZO-1 staining (as appropriate) We will visualise different channels in the barriers to document their potential for cell to cell signalling. We will examine the barriers with electron microscopy to check cell viability, number of cell layers, types of intercellular junctions and morphological responses to external stimuli. T2: Explore the mechanism and actions of the signalling.Taking DNA damage chromosomal aberrations and cytokine secretion as our endpoints, we will explore the role of connexins, pannexins and purinergic transmission in the primary cell barrier signalling. We will examine whether there may be distinct types of intercellular signalling pathways and mechanisms that lead to the secretion of DNA damaging molecules, molecules that cause tetraploidy or cytokines. We will examine more general responses of the target cells including apoptosis and altered cell division.

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We will use cobalt chrome nanoparticles as our exposing agent but will also explore different functionalities of the nanoparticles, whether coated with different protein coronas or with select modifications to their surface chemistry. This programme of work will explore the risks of nanoparticle exposure to a foetus, but more critically will examine the mechanism by which the risk might be incurred.T3: Use model predictions to derive design rules for the properties of nanoparticles (i.e., size, shape and charge) that optimize signaling across the barrier: Results from the barrier models will be communicated to partners NUID UCD, project ESR1 and 2, in order to correlate the signaling across the barrier with the nanoparticle cellular interaction and intracellular targeting of the nanoparticles within the barrier. Results -Deliverables:R1: PhD thesis R2: Report on mechanisms, kinetics and amounts of nanoparticle transport across foetal barrierR3: Report on nanoparticle-induced signaling across the foetal barrierDissemination: 1. Participation in three international conferences during M25 – M36.2. At least one research article to be published from M25 – M36. Planned secondment: ESR7 will have two, three-month secondments at NUID UCD and EPOS. At NUID UCD ESR7 will explore different functionalities of the nanoparticles, whether coated with different protein coronas or with select modifications to their surface chemistry. At EPOS ESR7 will complement the mathematical modelling with data from barrier signalling under different conditions, in order to provide predictions of signalling across barriers.Risk assessment: Nanoparticle signalling across barriers is now a well-established phenomenon in our laboratory and we have well established protocols in our laboratory to achieve this.More difficult and challenging is the establishment and creation of a more accurate model of the real placenta. There are challenges in maintaining a high level of cellular viability within the barrier. There is also a need to achieve a high level of syncytialisation and more specifically of the surface layer of the barrier. A variety of techniques will be used to overcome this challenge including the creation of barriers in two stages. Throughout this process we will compare results in BeWo cell barriers and primary trophoblast barriers, both of which ourlab can make as bilayed confluent barriers which signal.

FellowESR8

Host institution UNIMA

Duration36 months

Start dateM1

Project title: Design of degradable and targeted nanoparticle-based delivery vehicles with an emphasis on the brain tissueSupervisor name: Prof. Kostas Kostarelos (UNIMA) (main supervisor), and Prof. Jan Mollenhauer, University of Southern Denmark (SDU)PhD enrolment: Y

Objectives: O1: Fabrication of different types of nanoparticles of different shapes and chemical consistencies (month 12)O2: Physicochemical characterisation of different nanoparticles to be studied (month 24)O3: Cell internalization kinetics of the nanoparticles (month 36)O4: Tissue distribution (in vivo) and degradation kinetics (in vitro and in vivo) (month 42)

Tasks and methodology: T1: Engineering of nanocarriers (liposomal, nanotube, nanofiber, graphene) of controlled size (in the range 20-250 nm), shape (spherical or long axial ratios) and reference surface targeting grafted ligands (e.g. for brain, Apolipoprotein E)T2: Identification of key whole body-transport patterns, pathways and biological barriers (in particular in the brain)

Results: Deliverable 1 (D1): Engineering and characterisation of nanocarriersMilestone 1 (M1): Evaluation of the tissue distribution and residence kineticsDeliverable 2 (D2): Offer time- and space-resolved tracking of nanoparticles by imaging approaches Deliverable 3 (D3): Determine the clearance and degradation kinetics of nanoparticles from the brain tissue

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Dissemination: 1. Two publications in years 3 and 4 reporting Results above2. Presentation in two international conferences in the field of nanomedicine3. Contribution in schools/lectures for undergraduate students in year 44. Encourage communication in public/general media Planned secondment: S1: HZI, month 18-21 (3 months) to studying conjugation approaches of targeting ligands and in vitro investigations S2: ACS, month 33-35 (3 months) for development of cell-based assays to assess nanoparticle uptakeRisk assessment: No particular risks identified at the present time point.

FellowESR9

Host institution NUID UCD

Duration36 months

Start dateM2

Project title: Designing the bionanointerface for transcytosis and barrier crossing; WP1, WP3Supervisor name: Kenneth Dawson (NUID UCD); Co-supervisor: Kostas Kostarelos (UNIMA)PhD enrolment: Y

Objectives: Development of bionanointerfaces in which details of geometry of presentation of active sites are optimized toO1: minimize non-specific biomolecular bindingO2: optimize receptor engagement for endocytosisO3: optimize receptor engagement for transcytosis with a second targeting moiety

Tasks and methodology: T1: Synthesis of functional probe nanoparticles incorporating features optimised for spectroscopic detection and measurement through cell biology methods in fluorescence microscopy, flow cytometry, T2: Investigation of surface functionalisation chemistry to optimise for combined properties of dispersion stability, surface reactivity for biofunctionalisation and overall product functionality in complex environmentsT3: Introduction of combination orthogonal chemical functions at the surface for introduction of more than one biological function at separately addressable reaction points.T4: Exploration of this surface parameter space (tether lengths, tether densities, biological function combinations etc.) to find optimal interface for barrier negotiation.

Results: R1: Report on synthesis and characterisation of efficient, adapted for purpose nanoprobe materials, their surface functionalisation and their transfer into activities across WP1and WP2. R2: Report on exploration and optimization process applied to design and control of the bionanointerface working reciprocally with colleagues across WP1 and WP2 regarding incorporation of identified targeting moieties (ESR1) and building rationally upon initial cell/barrier interaction studies.R3: Ph D Thesis

Dissemination: 1. Participation in three international conferences during M28 – M42.2. At least one research article to be published from M35 to M42.Planned secondment: UNIMA: Training in design and surface modification of nanocarriers for interaction with biological barriers. 3 months in Year 1SDU: Training in design of biomimetic targeting moieties for nanoparticle targeting. 3 months in Year 2Genzyme: Industry secondmentRisk assessment: Naturally there is more risk in aiming for higher levels of innovation possible through collaboration across the program. For example, there is external dependence in the possible incorporation of synthesised novel targeting moieties (WP1, ESR8) and novel surface pattern motifs (WP1,2 ESR2). This project is designed however, such that the core work can be accomplished and envisaged to reach a proof-of-principle stage building upon previous investigations and expertise built up in the area of biofunctionalisation of nanoparticles, using available natural ligands and adapting developed surface functionalisation approaches.

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FellowER1

Host institution AvantiCell Science Ltd

Duration24 months

Start dateM7

Project title: Cell-Based Nanosafety Assay Design and Validation for Accreditation and DisseminationSupervisor name: Dr Colin WildePhD enrolment: NObjectives: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.Tasks and methodology: T1: Assay kits will be based upon primary human cells. ACS shall base initial “productisation” activity on systems incorporating hepatic Kupffer and renal stellate cells which are important barometers of hepatic and renal toxicity. These cells are relatively robust, and will provide an appropriate training experience for the ACS ER trainee; T2: 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; T3: Assay productisation shall also incorporate technology for cryopreserving cells in situ in multiwell plates, optimising reproducibility by minimising assay manipulations; T4: Kit development may use substrate-free reporting based upon primary cell-customised transfection technology, and the hands-free delivery of culture components by means of thermo-sensitive polymer carriers. T5: The ultimate objectives is to seek accreditation of a validated assay platform in kit form by the relevant European processes and to engage with industrial players with strong nanosafety interests e.g. the automotive industry.Results: The project shall: R1: produce cell-based assays capable of measuring nanosafety in a range of sample materials;R2: do so whilst meeting pre-determined quality criteria during beta-testing;R3: be capable of packaging into a prototypic product, in the form of a cell-based nanosafety test kit, which delivers end-user convenience and quality-assured gathering of nanosafety data. Dissemination: Dissemination of assay system capabilities via beta testing by end-users; marketing of service and products via international trade shows and commercial events.Planned secondment:NUID UCD (month12; duration 1 month; study of primary cell nanoparticle trafficking. EPOS-Iasis (month 18; duration 1 month; in vitro tissue reconstitution)Risk assessment: The project depends upon:• access to nano-engineered particles designed and produced by other Pathchooser project partners; ACS

shall liaise closely with project 9 partners (NUID UCD, UNIMA) for this purpose;• close relations with partners focused on developing mucosal epithelium cell-based systems e.g. partners

HZI, EPOS).

FellowER2

Host institution EPOS-Iasis, R&D

Duration24 Months

Start dateM12

Project title : Experimental testing of nanoparticulate systems for crossing intact and disrupted barriers (WP3,4) Supervisor name: Andreani Odysseos and Constantinos Pitris (EPOS) PhD enrolment: N

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Objectives:O1: To build advanced three-dimensional tissue constructs and tumor spheroids as enabling systems O2: To develop orthotopic animal models with intact and disrupted intestinal and brain barriersO3: To verify quantitatively the nanoparticulate interaction with model barriers O4: Specify and apply mathematical models to the delivery of nanoparticulate systems to normal and tumor brain and intestinal tissueTasks and methodology: T1: Construction of three-dimensional tumor constructs and spheroids mimicking the tumor intact and disrupted barriers: The constructs will be formed inside a capillary tube and the solution of nanoparticles will be placed in touch with the free surface of the construct. NPs will be equipped with a fluorescent moiety. Application of confocal microscopy and a proprietary algorithm in serial sections of the 3-dimensional phantoms is expected to allow for trafficking assessment across barriers and quantification of uptake. Data will be communicated to partners UNIMA and SDU for optimization of synthetic and grafting parameters. T2: Development of orthotopic animal models with intact and disrupted intestinal and brain barriers: In vivo models of tumor barriers will be developed from Glioma Stem Cell, which will be isolated as the CD133 subpopulation of primarily cultured glioblastomas spheroids and engrafted in immunodeficient NOD SCID mice through cranial window. For othrotopic intestinal models, Lgr5+ colon epithelial cells will be injected into the colon under fiberoptic endoscopic guidance and following trypsin exposure of the lumen. T3: Elucidation of nanoparticulate systems interaction with intact and diseased barriers in vivo: Selected nanoparticulate systems from partners UNIMA, SDU and MPI-CBG will be loaded with the indicated drug will be used for pharmacokinetic studies and barrier penetration. Tumors will be excised up to a boundary and nanoparticle/drug levels will be assessed. Areas immediately surrounding the core are expected to receive significantly less drug due to incomplete BBB breakdown. Peri-surgical in vivo molecular imaging will enable the detection of nanoparticle/drug uptake by distally engrafted glioma stem cells and recurrent tumor nests. Ex vivo studies will also be conducted on cryopreserved brain tissue of sacrificed animals. For intestinal tumors, penetration parameters will be compared between tumor and neighboring normal tissue. T4: Deciphering of molecular mechanisms and recognition patterns underlying transport in brain tumor barrier: With partner SDU, ER2 will study molecular transport systems at the interface between cellular transport and glioma carcinogenic pathways. Emphasis will be given on Caveolin-1, a major structural component of Caveolae, which are specialized plasma membrane invaginations involved in molecular transport, modulation of blood-brain barrier permeability and activation of major glioma pathways via EGFRvIII and Src.T5: Mathematical model validation in vivo: Simulations applied in IRP of ESR5 will be run to predict optimal nanoparticle properties that ensure effective penetration in both normal and diseased brain and intestinal barriers. Results from the stochastic models will be communicated to the consortium in order to reach consensus on optimal NP parameters and characteristics for normal vs disease barrier crossing.Results - Deliverables:R1: Two novel in vivo validation platforms for exploitation by other IRPs testing of NPs interactions with naturally occurring pattern recognition molecules in normal and diseased barriers (IPRs1,3 and 6-10)R2: Differential specifications of nanoparticulate systems for in vivo crossing in normal vs diseased barriers R3: Report on molecular clusters and/or pathways underlying NPs permeability across normal vs diseased barriersR4: Report on design rules for optimal penetration of NPs to normal vs diseased barriersDissemination:ER2 is expected to have at least I manuscript submitted and 1 manuscript in preparation by the completion of this fellow. Publications will be preceded by thoughtful evaluation of deliverables for patenting. Major international conferences on nanomedicine (e.g. ICoN2015, Euronanoforum ) will be highly sought for data presentation. ER2 will be strongly encouraged to participate with a PathChooser booth at the EC-supported “Scientists Night”.Planned secondment:From months 12 through month 14, ER2 will be seconded to KCL for training in techniques for glioblastoma sphere isolation in primary cultures and in in vitro models of transcytosis. From month 18-20 ER2 will be trained at SDU in advanced microscopy and imaging methods for endocytosis and deciphering of pattern recognition mechanisms via the Caveolin pathway.Risk assessment:Unavailability of adequate patient tissue: the partner has a good collection of cryopreserved glioblastoma stem cells and a collection of colorectal tissue as backup.High concentration of NPs in vital organs (liver, lungs): preliminary pharmacokinetic studies will be undertaken prior to functionalization of the particles in order to assess animals that would tolerate the treatment and NPs suitable for the study.

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A. Research MilestonesM1. Assessment of scientific progress. The PathChooser fellows will have weekly discussions with their tutors. Fellows will also deliver a progress report annually followed by an oral seminar and an interview with the second PI in charge of their training as defined by the Personal Training Plan (B.4.1). D5.1-3: Periodical scientific progress reports (Mo 18, 30, 42).

M2. Assessment of collaboration progress. ESR fellows will spend at least two months of their training in performing collaborative experiments, related to their personal research programme, in one or more of the other PathChooser institutions. They will give oral seminars as part of their visits. Annual reports will serve to assess progress. D5.4-6: Periodical collaboration progress reports (Mo 18, 30, 42).

M3. Fellows’ seminars. The PathChooser fellows are expected to give seminars at least twice a year at lab meetings, and at least once at one of the PathChooser meetings. D5.7-12: Periodical reports of fellows’ seminars (Mo 10, 16, 22, 28, 34, 40).

M4. Participation of fellows to international scientific congresses. Fellows are expected to participate in one international scientific workshop, summer school or congress per year (external to the PathChooser project). Examples of potentially relevant meetings include: Annual international Biological Barriers Conference and Workshop, Saarland University, Germany (early March each year) and the QNano Integrating conferences (February each year). Participation in top level meetings such as ESF / EMBL Conferences or Gordon Conferences on relevant topics will be encouraged. –D5.13-15: Periodical reports of fellows’ participation to congresses (Mo 15, 27, 39).

M5. Preparation of specialisation/PhD thesis. The PathChooser fellows are required to organise their research results in the form of a PhD thesis and to discuss them with the PathChooser Supervisory Board, before the official discussion at the competent University. Although the experimental work will be concluded at month 42, it is expected that the thesis will be discussed later.Note that fellows hired by industry partners will be enrolled at the coordinator institution, unless agreed with a more closely located academic partner within PathChooser. Indeed, a commitment to mutual recognition between NUID UCD and EPOS-Iasis is already in place and is included in the letters of support. D5.16: Fellows’ PhD thesis (Mo 48).

B.4.3. Management structure, organisation and proceduresIf necessary and upon approval by REA, the experts listed in this Description of Work might be replaced with scientists with equivalent expertise

B.4.3.1. Network organization and management structureAccording to the Special Clause 5 bis of Article 7 of the Grant Agreement, a mid-term review meeting must be organized, preferably during month 20-22 of the project, and all the fellows and beneficiaries need to be present. The venue and organization of this meeting will be of the responsibility of the coordinator, and the timing and location of the meeting must be agreed with the REA project officer

The coordinator of this project will be Prof. Kenneth A. Dawson who will be in charge of the scientific and technical supervision of PathChooser and will take responsibility for all the PathChooser financial and administrative issues on behalf of the participating institutions.

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Each partner institution’s principal investigator (PI) who will be responsible for research and training of the local fellows, and for training of visiting fellows based in other partner institution. Some PIs will act as Work Package (WP) leaders and deputy leaders, and be in charge of communication / public relations, gender aspects, monitoring of training and education, industrial contacts, and intellectual property issues. If a PI cannot participate in an official meeting another person from the same institution may be named as temporary substitute. All PIs and representatives from the associated level 2 partner are members of the Management Committee (MC), the body that will look after the coordination and management of the project itself, including technical and financial reporting, recruitment etc. The MC members are:1. Kenneth Dawson (NUID UCD)2. Jan Mollenhauer (SDU)3. Claus-Michael Lehr (HIPS)4. David Begley (KCL)5. Andreani Odysseos (EPOS)6. Marino Zerial (MPI-CBG)7. Patrick Case (UNIVBRIS)8. Kostas Kostarelos (UNIMA)9. Colin Wilde (ACS)10. Seng H. Cheng (Genzyme)11. Stavriana A. Kofteros (DIOGENES)

Composition of supervisory board (including involvement of the private sector)A Supervisory Board (SB) consisting of all MC members plus outstanding representatives from academic institutions, industry and regulatory agencies will provide external input and monitor the progress of the training and research programs. Proposed external members of the SB are:

1. Ysbrand Poortman, International Genetic Alliance /Patients Network for Research in Health2. Maria Papaluca, Director European Medicines Agency3. Joseph Brain, Harvard School of Public Health4. Jörg Kreuter, Goethe University5. Kyle Landskroner, Senior Lab Head, Preclinical Pharmacokinetics and Metabolism, Actelion

Pharmaceuticals Ltd., Switzerland.

As described in Section B3, a representative of the ESR/ER Fellows sit on the Supervisory Board, to ensure that the fellows are also involved in the planning and implementation of their training. All ESR/ER will be involved in organisation of the various workshops and meetings, especially those being hosted by their local institution.

The SB will provide external evaluation, monitoring and advice. It will review and agree the individual training and career plans of each fellow, monitor the progress of the fellows and of the research projects, advice on ethical questions and on strategies both during the running time of the ITN and beyond, and advice and monitor gender equality and communication activities.

The coordinator will act as contact point between the WP leaders, the Management Committee, the Supervisory Board and the Commission. He will have supervisory responsibility for monitoring progress in scientific and training activities. In the case of problems or delays he will immediately communicate with the EU Commission in order to ensure that corrective actions are agreed upon and implemented at an early stage. The coordinator will arrange timely meetings both of the Management Committee and of the Supervisory Board. Regarding financial and administrative issues, the coordinator will be supported by a dedicated assistant and by the administrative and legal staff of NUID UCD

The Management Committee (MC) will be the general assembly of all partners, represented by their PIs. Key decisions with respect to milestones, deliverables, work plan etc. will be made in the MC by majority vote; however, the coordinator will make every effort to reach consensus decisions.

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Communication: Two MC members (J. Mollenhauer and K. Dawson) will be responsible for communication within and from the project using Web-based technologies. These MC members will also be in charge of public relations. They will establish a web site (based in Dublin) which will be partially accessible exclusively as an intranet platform of PathChooser and as a fellows’ virtual meeting room, but will also serve as general information site for the public. This site will also be used to disseminate information about experimental problems and limitations encountered during the individual projects. Communication of project results with all related stakeholders, such as industry, regulators and policy makers, and the general public will also a task of the communication team. All fellows will be encouraged to participate in science outreach events nationally, and internationally, such as Science Weeks, City of Science events etc.

Two MC members (A. Odysseos and C.-M Lehr) will be responsible – in close cooperation with the coordinator - for gender aspects.

Two MC members (D. Begley and A. Odysseos) will address training and education aspects, monitor local training activities regarding scientific and complementary skills, and supervise the secondments and visits of the trainees. They will also organize network wide training activities like “theory seminars” and “laboratory seminars” in the various partner institutions, together with the local hosts and the coordinator. At the start of the project they will produce a Manual for Fellows which will be distributed to all fellows at the start of employment and which will explain in detail the ITN and its procedures, good scientific practice, job perspectives and other relevant issues. The representatives of industrial partners (C. Wilde, A. Odysseos and S. Cheng) will jointly report to the management committee on the industrial relevance of the training activities. This will ensure that the trainees acquire a wide range of skills needed in the present job market.

Three MC members (K. Dawson, C. Wilde, and A. Odysseos) will be contact persons for intellectual property issues and will ensure best practice as well as adherence to the rules set up in the Consortium Agreement. C. Wilde and A. Odysseos represent SMEs with significant patent holdings and interests. M. Voetz represents an industrial partner that is a capable supplier of technology solutions for the chemical and pharmaceutical industries. These partners will oversee an IPR trainingcourse set up for PathChooser fellows which could include a visit to the European Patent Office at Munich with presentations of patent experts.

The four work packages will be headed by work package leaders, who will monitor the progress of the WP (work schedule, milestones, tasks, and deliverables), facilitate WP communication and provide reports in time. At the MC meetings, WP leaders will report on progress, problems and outlook of their WP. Designated WP leaders are indicated in Table B.2.1.1.

Rules for decision making and conflict resolutionA Consortium Agreement will be signed by all partners of the ITN in order to set out in writing the responsibilities and management structure of the ITN and the fair participation of all teams.

B.4.3.2. Financial managementThe management budget for the consortium will be largely maintained by partner 1 (NUID UCD), and from this travel and meetings will be funded. Partner 1 will also distribute budgets as agreed, according to the guidelines laid down by Commission.

B.4.3.3. Recruitment strategyRecruitment: Fellows will be recruited in agreement with The European Charter for Researchers, in particular the Code of Conduct for the Recruitment of Researchers. Positions will be announced using all reasonable instruments, like print media, posters, flyers and electronic platforms, including the EC job database (http://ec.europa.eu/euraxess/jobs), which also advertises via Nature jobs. Effort will be to ensure the best students from less favoured regions are informed.

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The criteria of selection, in particular for ESR, will be the quality of the candidates Studies + curriculum vitae, their expressed interest in research (via their letter of application) and the expected benefit of the ITN training for the candidate (also outlined in their letter of application). The selection criteria will be published in the calls, to ensure full transparency of the recruitment process. All partners will implement the tenets of the European Charter for Researchers and the Code of Conduct for the Recruitment of Researchers, and will ensure that the entire recruitment process is conducted within the scope of this charter.

In the unlikely event that a fellow is not recruited through a round of interviews, another round of recruitment will be opened. If, following this there is no successful candidate the management board will be notified and appropriate action will be taken.

B.4.3.4. Gender aspectsThe PathChooser ITN will strive to establish and maintain a gender balance, which is in nanosciences and nanomedicine is a particularly important task since technical disciplines are still far from gender equality. High quality training and network building for young female scientists will be a particular focus.

B.4.3.5. Consortium Agreement A Consortium Agreement will be signed by the network and submitted to the REA within the first 2months of the project.

B.4.4. Project monitoring and key performance indicators

Periodic and final reports are contractual deliverables, according to Article 4 and II.4 of the Grant Agreement. In addition, progress reports are due at the end of the first and third year in order for the REA to monitor the implementation of the project. The reports will include the information below.

B.4.4.1. Research Activities; Research results obtained (including a short description of progress on the individual projects) and deviations, if any, to the original research work plan.Scientific highlights and achievements (scientific/technological breakthrough, patents, awards, prizes etc...). A full list of individual and joint publications, directly related to the work undertaken within the project (including citation index and impact factor), with appropriate acknowledgment of the funding source. Intersectoral and multidisciplinary collaboration.

B.4.4.2. Training ActivitiesImplemented training events/activities and deviations, if any, to the original training plan (including Career Development Plans, coaching or mentoring activities in place at each host institution).Participation of the fellows in training events and meetings from the network (workshops, seminars, summer schools, etc), and at international conferences outside the network (names, places, dates).Transferable skills training (e.g. project management, presentation skills, language courses, ethics, intellectual property rights, communication, entrepreneurship, etc.). Implementation of visits/secondments undertaken within the network to both full participants and associated partners.

B.4.4.3. Management activitiesStatus of ESR/ER recruitments at each participant, and relevant issues related to the recruitment strategy/process and gender balance, with justification for any deviation from the original plan.Effectiveness of networking, communication and decision-making between stakeholders.Effectiveness of the "training events and conferences": external participation and integration in the training programme.Effectiveness of the financial management and compliance with Marie Curie salary rates. IPR management and commercial exploitation of research results.

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B.4.4.4. Dissemination and outreach activitiesImplementation and analysis of the proposed outreach activities and deviations, if any, to the original work plan. Analysis of the dissemination activities.

B.5 Impact

B.5.1. Impact towards the policy objectives of the programme

Contribution of the PathChooser training programme to improving career prospects and employability of researchers, including ERs where appropriate

A crucial aspect of this will be ensuring that the young researchers are trained to work under the conditions of open innovation and with a deep understanding of the commercial realities faced by industry. Thus (as per the European Commission / ETP Nanomedicine Expert report 2009) expensive delivery technologies will not be commercially viable unless the pharmaceutical contribution is low cost and/or there is significant targeting of the device to a specific target leision.24 By including awareness of commercial reality and open innovation into the curriculum and training plan of the PathChooser Fellows, the programme will ensure that the fellows are fully employable by, and indeed highly desirable to, potential employers.

The Personal Training Plan to be set up for each fellow will address career planning at an individual level. The fellows will acquire in-depth knowledge about an emerging inter/multidisciplinary field, where they will gain experience with academic as well as with industrial partners, leading to awareness of the inter-sectoral approaches necessary for success in delivery across biological barriers. The training programme will stress, for ER particularly, management skills, regulatoryaspects, IP and open innovation considerations.

Contribution of the PathChooser training programme to stimulating creativity and entrepreneurial mind-set of researchers at doctoral level

The scientific breadth of the work plan, the state-of-the-art and cutting edge methodologies involved, the content and quality of the training program emphasizing entrepreneurial skills and the human resources dimension of the research study will provide the background and opportunities to stimulate creativity and an entrepreneurial mindset in the young researchers, as they learn to effectively compete for these resources and establish their own independent laboratories and further move on the establishment of their own start-ups.

Contribution of the training programme to the policy objective of structuring the initial research training capacity at the European level (through establishing longer term collaborations and /or lasting structured training programmes between the partners' organisations)This field requires international collaborations between research institutes, universities and industries in order to define standards and to structure the large amount of information in this field, and to enable truly innovative approaches to emerge. PathChooser addresses these issues by creating an interdisciplinary and intersectoral training environment for young researchers, who will not only obtain experience in hands-on science, but also become part of a network of peers and experienced scientists.

In the long term, the partnership aims at setting up the foundation for lasting technology training capacities at the European level. As a first step, it is envisioned to apply for an Erasmus Mundus cooperation and mobility program in relation to Nanobiotechnology in Drug Delivery. Collaboration

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with the ongoing Innovative Medicines Initiative (IMI) program for Education will be pursued. The training modules of the ITN will be exploited by partners to create a cluster of Industrial Doctorates in subsequent Marie Curie calls.

The contribution of the training programme towards the policy objective of enhancing public-private sector collaborations in terms of research training;

This inter-sectoral mobility program will provide researchers many opportunities to acquire skills which are equally relevant to the public and private sectors. As explicitly outlined in the “transfer of knowledge” section, this not only encompasses the new technical skills and competencies that will be acquired but also includes the development of the seconded researchers in many other ways. In particular, the researchers will be involved in the transfer, teaching and establishment of new technology across sectors and face the challenge of discrepancies between academic and entrepreneurial culture and research outlook. They will be involved in project management, the co-organisation of seminars and international conferences, and the responsibility for important laboratory equipment and exposure to a wealth of opportunities for complementary skills bridging the two skills required by the sectors, thus having an unprecedented opportunity to become future leaders in either sector.

B.5.2. Plans for exploitation of results and Dissemination strategyThe training events which are outlined in section 3.1 are closely related to the dissemination strategy.

A website will be developed and created by the fellows; this is an important part of the dissemination strategy. Not only does it allow for peer-to-peer communication and learning, but it allows the fellows to disseminate scientific findings to the scientific community, industry and general public. The website will be regularly updated and maintained for the life of the project.

Two international conferences will be organised by the fellows; 1st conference: In vitro-in vivo correlation in models of biological barriers, 2nd conference: Designing the bio-nano-interface for drug-delivery carriers. It is required that all fellows disseminate their results at both of these conferences, giving the opportunity to disseminate to the scientific community.

As part of one of the 6 monthly meetings, a Meet-the-Manager Workshop will be arranged. As part of this workshop all fellows will disseminate their findings to the manager. This activity will encourage dissemination to industry.

In addition to the above dissemination plans, each supervisor has included a dissemination activity under the fellow description, this includes conference attendance, paper publication, attend workshops, attend tradeshows and organise a PathChooser booth at the EC-supported “Scientists Night”.Plans for exploitation of results emerging from the PathChooser projectThe research outcomes will initially be documented for intellectual property disclosure to the Partners and for patenting. The processing of any patent application will be full responsibility of the partner owning the relevant IPR. However, for the exploitation of the patents, SME partners will have the right of first refusal.

Special care will be taken to ensure that patenting does not interfere with ESR fellows progression towards their PhD, and thus and accelerated process will be applied where IP arises. We anticipate conceding the rights to any licensees arising from the research in the beginning of the 3rd year following completion of the project, to allow for patent filing, negotiation of rights etc.

As required by Annex II of the grant agreement, the coordinator should ensure that all publications and presentations by members of the project consortium - including all funded fellows - acknowledge the EU financial support received. This acknowledgement should specifically refer to the Marie Curie Initial Training Networks (ITN) action, as well as the project number and acronym.

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B.5.3. Outreach activities

Outreach entails communication initiatives directed to the general public, rather than to the research community or other stakeholders. The overall goal of this series of activities is to create awareness among the general public about the research work performed in the PathChooser ITN and its implications for citizens. As well as raising the profile of MCA with the general public, outreach activities will also introduce students from schools and universities to science, research and innovation. These students will be encouraged and motivated to pursue careers in science. The PathChooser Fellows will be encouraged to develop the following types of outreach activities:

A: Clustering with EC-FP7-funded projects on communicating nanotechnologies and related actions such as EC Nano Communicating Roadmap. Fellows, under the supervision of the supervisors and management team, will be encouraged to host events, which are combined with the Complementary Skills courses, but will include additional actives:

- Debating ethical, legal and social aspects of nanobiotechnology and nanomedicine- Awareness-raising and outreach on the topic of nanotechnology for drug delivery- Nano-schools: pedagogy, teacher training and teacher training material.

B: A public-awareness event for non-medical professionals the margins of the international conference: In the margins of the international conferences planned (London and Cyprus, months 18 and 36, respectively) a satellite event will be organised by the fellows for the general public with immediate translation and concurrent broadcast on local radio. The tentative topic would be “Targeted therapy with Nanodrugs– Nano or Mega hopes?”. The objective would be to sensitize patient organisations and lay individuals about the special opportunities and challenges around nanomedicines. The event will be interactive and form a public debate. Politicians and activists will be invited. This event will be organised by the fellows, with the support of the supervisor and the management team.

C: Participation of ESRs/ERs in the “ACS Nanotation Nanotube” contest: This is a video contest for explaining nanoscience and nanotechnology to public. The video will be judged by an ACS panel based on creativity, scientific, clarity of explanation, originality and overall quality as well as by popular choice. The PathChooser ERs/ESRs will be strongly encouraged to participate on behalf of the network.

Participation of ERs in the “Fame-Lab –Talking Science” contest: This event is now established in UK and in Cyprus and expanding rapidly to other EU countries, attracting international participants, and the partners will make effort to set the bases for other participating countries. The Concept of the contest is to promote the talent of young scientists in communicating science. The aim is to encourage scientists to inspire and excite public imagination with a vision of science in the 21st century. The ERs/ESRs participating in PathChooser will be strongly encouraged to participate on behalf of the network.

D: The “Researcher’s evening”: This is a well-established activity organised by all member states funding organisations on the exact same day (Friday of third week of September) where research organisations or teams are invited to demonstrate their activities to the public in an inspiring and celebrating atmosphere. PathChooser partners (supervisors and fellows) will seek active representation with their own booth and demonstration material.

E: Newspaper articles: partners of PathChooser are already very active in delivering the nanomedicine message to the public through local and international newspaper and magazines. The actual topic of this ITN is of major significance for public health and ESRs will be strongly encouraged and assisted during Complementary Skills courses to write articles for public use.

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Each fellow will be involved in at least one outreach activity every year

For outreach activities A, C and E (described above) the outputs will be physical materials (i.e. webcontent, short articles, protocols for simple laboratory experiments etc.) targeted to the general audience. These materials will be made available through the PathChooser website (outreach section) and via other educational portals, such as the QNano Knowledge Hub. The materials will also be distributed via mailshots (e.g. MailChimp) to relevant stakeholders. For all webpages / weblinks and mailshots, Google Analytics will be used to monitor the number of hits on the various webpages, the number of downloads of specific content and the number of emails opened and further circulated for the mailshots. This will give is a good estimate of the success of the dissemination of the materials, and these approaches are already working well within the QNano Research Infrastructure (coordinated by NUID UCD). Additionally, all of the materials prepared will be published as Open Access documents under the terms of the Creative Commons Attribution License. This is an Open Access document distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

For outreach activities B and D, direct interaction with the public is envisaged, which allows a more direct monitoring of the impact and usefulness of the outreach activities. Here we propose to ask participants to fill out a short questionnaire about their understanding of nanomedicine before and after the event, as well as asking participants to provide any suggestions they have for increasing public awareness of nanomedicine and the project in general. The summer school participants may also be asked to present what they have learned about NanoMedicine during the summer-school, to help us to assess the success of the initiative. Suggestions taken during the first conference will be used to improve the subsequent event.

The outreach material developed for outreach will become part of the partners repository of public outreach material, and may be used at future open-days, or other public engagement activities by the partners, thereby further promoting the PathChooser ITN and the Marie Curie Actions

B.6 Ethical issuesThe Beneficiaries accept to uphold the highest standards of scientific integrity and ethical conduct during the implementation of the grant agreement. Ethical standards and guidelines compatible with, and equivalent to, those of FP7 will be rigorously applied, regardless of the country in which the research is carried out.

The project outlined here will strictly comply with widely recognized international texts and codes of practice, including the Helsinki Declaration and the Convention of the Council of Europe on Human Rights and Biomedicine. In addition, the project will be approved by the NUID UCD Ethics Committee prior to commencement, and should any specific ethical requirements be required (beyond those outlines below) these will also be obtained prior to commencement of the relevant research.

The applicants are aware of the potential danger from the interaction of nanoparticles with living systems. All partners and all recruited ESR and ER will treat relevant nanoparticles - as long as not proven otherwise - as potentially hazardous materials to humans and accordingly take appropriate precautions and safety measures to protect themselves, their colleagues and the environment, in accordance with the protocols implemented within the Centre for BioNano Interactions, and with the EU Code of Conduct on responsible nanotechnologies.

Copies of ethical approvals by the competent legal local/national Ethics Boards/ Bodies/ administrations will be submitted to the European Commission prior to the commencement of the research. If there is transfer of human biological samples and data between partners and any third parties, ethical approval will be applied for and submitted.

An external independent Ethics Advisor; Gunter Oberdorster will be appointed to oversee the ethical concerns involved in this research. A report by the Ethics Advisor will be submitted to the European Commission with the Periodic Reports.

This PathChooser ITN Research Training project does not a) deal with human cloning for reproductive purposesb) involve modifying genetic heritagec) involve children or others unable to give consente) involve use of non-human primates.

EU legislationThe project will respect all ethical requirements in its objectives and methodology. We will strictly comply with widely recognized international texts and codes of practice, including the following:

• Helsinki Declaration in its latest version • Convention of the Council of Europe on Human Rights and Biomedicine signed in Oviedo on

4 April 1997, and the Additional Protocol on the Prohibition of Cloning Human Beings signed in Paris on 12 January 1998

• UN Convention on the Rights of the Child* • Universal Declaration on the human genome and human rights adopted by UNESCO

Ethics trainingAccording to the Ethical guidelines, ethical training for all the ESR/ER involved in the project will be provided, this is compulsory. Ethics training will take place in all of the common training sessions. It is proposed that the centrepiece will include Course 1; PhD Induction Program and Knowledge Protection (SDU and NUID UCD, 5 days, month 7, or equivalent) and will focus on the Ethics and Use of Human tissue and Animal Models. This will ensure that all fellows are aware of the ethical issues.

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Research on Human Embryo/ Foetus

UNIVBRIS have good access human placenta in their hospital based laboratory and will therefore be able to compare the structure and the signalling of these model cell barriers with the real placenta. Full ethical approval for this work is already in place in Bristol.

The stem cells that will be used in the research project of ESR7 are obtained from recognized cell banks including the MRC. These are a national resource.

Research on Humans / Human Biological samples

When submitting the application for scrutiny to the competent local/national ethical boards/bodies for authorization/opinions/notifications, detailed information will be provided on the informed consent procedures that will be implemented. Example copies of the Informed Consent Forms and Information Sheets will be included. These will be in language and terms understandable to the participants. Participants must have the right: a. To know that participation is voluntary b. To ask questions and receive understandable answers before making a decision c. To know the degree of risk and burden involved in participation d. To know who will benefit from participation e. To know the procedures that will be implemented in the case of incidental findings f. To receive assurances that appropriate insurance cover is in place g. To know how their data will be collected, protected during the project and either destroyed or reused at the end of the research, h. participants should be duly informed, and consented also for this further usage, i. To withdraw themselves and their data from the project at any time j. To know of any potential commercial exploitation of the research.

Confirmation will be provided outlining that all the human samples used in this project are either legitimately available commercially or have been obtained following appropriate ethical approval.

Applicants will obtain from the data controller of their institution, written confirmations for the technical data protection procedures (data collection, storage, protection, retention and destruction and confirmation that they comply with national and EU legislation) and privacy/confidentiality measures that will be implemented in the project. These confirmations will demonstrate compliance of the data protection processes with the European legal framework. Copies of these confirmations will be forwarded to the European Commission prior to the commencement of the related studies. If requested by their institution’s data controller, applicants must consider obtaining approvals/opinions/authorizations from their national data protection authorities for the intended data collection and processing (http://ec.europa.eu/justice/policies/privacy/docs/wpdocs/others/2006-07-03-vademecum.doc). If requested, copies of these documents will also be provided to the European Commission. In cases where the host institution does not have a dedicated data protection officer, and only then, the applicant must contact the national/regional data protection authorities.

NUID UCD

The proposed projects to be undertaken in NUID UCD may use biological samples such as human serum or plasma for selected experiments. Human plasma is obtained from the Irish Blood Transfusion Service, in St. James hospital in Dublin (no medical data are associated with the samples, and donors are fully anonymous) and it is used in line with NUID UCD best practice and ethical guidance on handling of human biological materials. Typically, blood samples from 6 healthy volunteers (aged 25-55) are pooled for these studies, and no information regarding their identity is provided with the samples, ensuring their anonymity.

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Full ethical approval for all studies of the interaction of nanoparticles with human plasma has been obtained from the NUID UCD Research Ethics Committee (http://www.ucd.ie/researchethics/).

No personal data is supplied with the blood samples. No additional consent is required from the donors (consent is given to the Irish Blood Transfusion Board for multiple uses), and no information is returned to them regarding the outputs of the project. Plasma is isolated according to HUPO guidelines, and samples are stored at -80 °C until use. Samples are only frozen and thawed once.

Research on AnimalsAnimal studies are central to this work in order to validate the in vitro models to assess the efficacy of the various targeting approaches to delivery therapeutics across biological barriers. In order to minimise the number of animal experiments required the partnership will evaluate promising targeted moieties in tissue phantoms/constructs initially, and will apply animal experimentation only after the described mathematical model is validated in those phantoms.

Research will be carried out in compliance with revised Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes

Copies of ethical approvals by the competent local/national ethical/legal bodies, together with copies of relevant authorizations for animal experiments will be forwarded to the European Commission prior to the commencement of the research.

When submitting the application for scrutiny to the competent local/national ethical boards/bodies for authorization, detailed information should be provided on why living animals have to be used and why that species has been chosen. In addition, information should be given on the numbers of animals to be used in experiments, the nature of the experiments, the procedures that will be carried out and their anticipated impact (e.g. potential for pain, suffering, distress) and how that has been minimised. Furthermore, details should be provided on what procedures have been implemented to ensure the welfare of the animals during their lives (e.g. husbandry, minimising harms, criteria for humane endpoints, inspection protocols). The applicant should provide evidence of awareness of relevant European legislation and regulations covering animal experimentation and that the Principle of the Three Rs will be rigorously applied. (see below for further details).

Distress: In order to determine responsible endpoints, one must be aware of the general signs of distress. Distress should be taken to mean stress, discomfort, agitation, fear and pain. These symptoms are accompanied by changes in the physiology and biochemistry of the animal and can lead to pathological changes. There are no objective criteria for measuring the severity of distress. It is always a subjective assessment made by one or more people. Assessing the level of distress will always be a result of continued exchanges of opinions between the researcher, the laboratory animal expert and bio-technician, particularly in the case of new experimental situations. Appropriate measures will have to be taken on the basis of consultation.

Signs of distress: With respect to signs of distress, a distinction can be made between general key signs of moderate to serious distress on the one hand, and species-specific and model-specific signs on the other.

Examples of general key signs of distress include: -altered eating and drinking habits; abnormal growth or drop in bodyweight; abnormal body temperature; altered consistency, colour or amount of faeces; lack of inquisitive behaviour and isolation; abnormal posture and locomotion; altered depth and frequency of breathing; abnormal reactions to external stimuli.

MPI-CBGThe experiments described in this proposal will require the use of laboratory mice (C57BL/6), mus musculus, a commonly used inbred strain for mice studies. The use of mice in this project is based on

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the fact that they represent an established in vivo model system for studying intracellular trafficking on the tissue and organ level. Moreover, the already available procedures for the isolation of primary cells and tissues make the use of the material in the proposed experiments easier and the extrapolated data much more precise (as primary cells represent a much more accurate phenotype than any available cell-line). Finally, since this project aims at developing novel strategies for the delivery of siRNAs into cells for therapeutic applications, it would benefit from the use of a mouse model system.

Mice will be used for the following procedures:

1. In vivo RNAi delivery to the liver through tail vein injection2. Scientific sacrifice of mice (for i.e. organ collection)3. Isolation of mouse liver tissue and mouse hepatocytes after RNAi-mediated treatment

The number of mice that will be used is reduced to 1-2 mice per experiment (in total, the project will require around 150 animals).For the RNAi delivery studies, the mice will be injected with lipid nanoparticles encapsulating siRNA complexes via tail vein injection. These injections will be carried out by trained and experienced technicians at the animal facility in the Max Planck Institute of Molecular Cell Biology and Genetics. This way the animals will be exposed to minimal distress or pain. Furthermore, the lipid nanoparticles have no reported toxic side-effects at the used concentration range. For the isolation of liver tissue and hepatocytes, the mice will be fully anesthetized. The extraction procedure requires the perfusion of the liver during which the animal is killed. In these experiments, the animals will not be exposed before to other treatments, therefore no pain, suffering, distress or lasting-harm, other than the sacrifice of the mice, is inflicted. The animals will be kept in good conditions provided by the animal facility of the Max Planck Institute of Molecular Cell Biology and Genetics. The animals will receive ab libitum feeding and constant watersupply, their cages will be enriched and individually ventilated to ensure the welfare and good health of the animals. The feeding, breeding and cleaning will be performed by trained animal caretakers of the animal facility.All animal studies will be conducted in accordance with German animal welfare legislation and in strict pathogen-free conditions in the animal facility of the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Protocols were approved by the Institutional Animal Welfare Officer (Tierschutzbeauftragter) and all necessary licenses were obtained from the regional Ethical Commission for Animal Experimentation of Dresden, Germany (Tierversuchskommission, Landesdirektion Dresden).

EPOS-IASISAnimal studies are central to this work in order to assure effective delivery of Nanoparticles and Carbon Nanotubes into tissues. In order to minimize the number of animal experiments required the partnership will have already evaluated these promising compounds in tissue phantoms/ constructs and apply animal experimentation only after the described mathematical model is validated in those phantoms.

Distress: In order to determine responsible endpoints, one must be aware of the general signs of distress. Distress should be taken to mean stress, discomfort, agitation, fear and pain. These symptoms are accompanied by changes in the physiology and biochemistry of the animal and can lead to pathological changes. There are no objective criteria for measuring the severity of distress. It is always a subjective assessment made by one or more people. Assessing the level of distress will always be a result of continued exchanges of opinions between the researcher, the laboratory animal expert and bio-technician, particularly in the case of new experimental situations. Appropriate measures will have to be taken on the basis of consultation.

Signs of distress: With respect to signs of distress, a distinction can be made between general key signs of moderate to serious distress on the one hand, and species-specific and model-specific signs on

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the other. Examples of general key signs of distress include: -altered eating and drinking habits;abnormal growth or drop in bodyweight; abnormal body temperature; altered consistency, colour or amount of feces; lack of inquisitive behaviour and isolation; abnormal posture and locomotion; altered depth and frequency of breathing; abnormal reactions to external stimuli. For EPOS, based at the Republic of Cyprus, all animals are housed in accordance to the European Legal framework existing for the Protection of animals used for experimental and other scientific purposes (European Convention ETS 123 and Directive 86/609/EEC) as well as the current Guidelines of International Organizations such as the Association for the Assessment and Accreditation of Laboratory Animal Care International-AAALAC Int., and the Federation of European Laboratory Animal Science Associations-FELASA. Transgenic mice are housed under SPF conditions, in Individual Ventilated Cages. All the electromechanical equipment of the facility is monitored by a Building Managing System (BMS) 24 hours per day. The Animal Facility implements a complete veterinary medical care programme which includes preventive medicine, surveillance, diagnosis, treatment and control of diseases as well as veterinary care of the animals used in experimental protocols. A health monitoringprogramme is also in force, in accordance to the Guidelines issued by the Federation of European Laboratory Animal Science Associations.Application of the 3Rs will be demonstrated by the use of multiple live small animal imaging to reduce the total of number of animals used, when applicable. The animals are going to be imaged non-invasively, so it allows animals to be humanely sacrificed before significant adverse effects became evident. Furthermore, the image approach also allows the disease to be tracked over time in individual animals as their control allowing a significant reduction of the number of animals needed for all ourexperiments.

The 3R’s concept: All animal experimentations will be performed in accordance to the 3R’sPrinciples of Human Experimental Technique of Russel and Burch. During the drafting stage of the in vivo protocols a biostatisticial analysis (power analysis) has been performed in order to define the ideal number of animals, which will be used in conjunction with the creditability of the obtained results. A detailed study of the available in vivo experience and a systematic review and meta-analysis of the existing research literature will be performed in order to avoid additional animal experimentations and to further reduce the number of animals used. For each series of experiments pertaining to each compound with promising biocompatibility, bioavailability, delivery and efficacy properties the partnership will use the smaller number of animals required in order to achieve statistically meaningful data. Based on previous studies, this is 12 animals/group (i.e. untreated, positive control, negative controls) for each selected compound. All animal experimentations will be performed under the supervision of a laboratory animal veterinarian for the early recognition of any welfare or distress signs as well as for the early recognition of the humane end point. Euthanasia of the animals will be performed according to the European Commission’s and to the Federation of European Laboratory Animal Science Associations’ (FELASA) guidelines (reference: Laboratory Animal (1996) 30:293- 316, Laboratory Animal (1997) 31:1-32). Special concern will be given to the early estimation of the human end point based on the OECD guidelines. The development of orthotopic brain tumors will be performed in NOD/SCID mice. All experimental procedures will be performed aseptically under general anesthesia by using intravenous anesthetic agent (Avestin). 30 minutes before surgery an analgesic agent (caprofen) will be administered to the animals. Analgesic treatment will be repeated also three hours after the operation and after that every eight hours for the following two days. During that time animals will be under veterinary care for early recognition of any pain and distress. During the two following the operation days a special gel formula (Solid Drink®) will be provided to the animals for hydration and feed. Pain can have negative effect on the immune system, thereby accelerating the growth rate of the tumour. Thus pain relief may influence the results of the experiment.

All animals will be housed, cared and used in accordance to the Commission Recommendation 2007/526 and the recently published European Directive 2010/63.

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UNIMAFor animal studies at Partner 8, any experimental procedure involving animals has been described and authorised by the UK Home Office (Project License no.: PPL/70/5910) and approved by the University of London ethical committees in compliance with the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Application of the 3Rs will be demonstrated by the use of multiple live small animal imaging to reduce the total of number of animals used, when applicable.

The animals are going to be imaged non-invasively, so it allows animals to be humanely sacrificed before significant adverse effects became evident. Furthermore, the image approach also allows thedisease to be tracked over time in individual animals as their control allowing a significant reduction of the number of animals needed for all our experiments.

For animal studies at UNIMA, any experimental procedure involving animals has been described and authorised by the UK Home Office (Project License applied for) following approval by the University of Manchester ethical committees in compliance with the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Application of the 3Rs will be demonstrated by the use of multiple live small animal imaging to reduce the total of number of animals used, when applicable.

Compliance with the 3Rs policy

The 3Rs concept: All animal experimentations will be performed in accordance to the 3R’s Principles of Human Experimental Technique of Russel and Burch. During the drafting stage of the in vivo protocols a biostatistical analysis (power analysis) has been performed in order to define the ideal number of animals, which will be used in conjunction with the creditability of the obtained results. A detailed study of the available in vivo experience and a systematic review and meta-analysis of the existing research literature will be performed in order to avoid additional animal experimentations and to further reduce the number of animals used. For each series of experiments pertaining to each compound with promising biocompatibility, bioavailability, delivery and efficacy properties the partnership will use the smaller number of animals required in order to achieve statistically meaningful data. Based on previous studies, this is 12 animals/group (i.e. untreated, positive control, negative controls) for each selected compound.

All animal experimentations will be performed under the supervision of a laboratory animal veterinarian for the early recognition of any welfare or distress signs as well as for the early recognition of the humane end point. Euthanasia of the animals will be performed according to the European Commission’s and to the Federation of European Laboratory Animal Science Associations’ (FELASA) guidelines (reference: Laboratory Animal (1996) 30:293-316, Laboratory Animal (1997) 31:1-32). Special concern will be given to the early estimation of the human end point based on the OECD guidelines.

Dual use

In designing strategies to target biological macromolecules to cross biological barriers, there is always the potential for mis-use of this knowledge by others for harm, such as the development of biological weapons utilising the targeting strategies developed within the project. Every effort will be made to ensure responsible use of the data generated, and that the targeting strategies developed are unlikely to exploitable for harm. Consideration of this aspect will be included in the project risk register which will be utilised to monitor the potential risks to and from the project’s success.

In the event that dual use is discovered, all relevant parties within the research institution will be notified along with the commission. The potential dual use implication will be examined and reported upon.

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PART C:

Overall indicative project deliverables

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PART D:

Overall maximum EU contribution

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Appendix 1: Gantt chartMonths 1 2 3 4 5 6 7 8 9 1

011

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42 43 4

445

46

47

48

WP1-4(research)

ESR1ESR2ESR3ESR4ESR5ESR6ESR7ESR8ESR9ER1ER2

WP5(training)

Workshops

Conferences

WP6(diss. & outreach)

Dissem.

WP7(mngt.) Meetings K

O

FKO

PM

PM E

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Appendix 2: Extract from the 2013 PEOPLE Work Programme

Structure of the cost categories applicable for ITN (adapted from Table 3.1 and 3.3 of the WP)This information does not substitute the relevant information of the 2013 People Work Programme, which should be consulted for further details.

1Monthly living

allowance

2Monthly

mobility allowance

3Contribution to the training expenses of eligible researchers

and research/transfer of knowledge programme expenses

4Management activities

(including audit certification if applicable)

5Contribution to overheads

Flat rate of :

38 000 Euro/year for ESRs and 58 500 Euro/year for ERs

Rate for individual countries is obtained by applying the correction coefficients listed in Table 3.2 of the WP.

Flat rate allowance to cover expenses linked to the personal household, relocation and travel expenses of the researcher and her/his family in the host country: reference rate of EUR 700 for researchers without a family and EUR 1000 for researchers with a family.

Rate for individual countries is obtained by applying the correction coefficients listed in Table 3.2 of the WP.

For multi-partner ITNs and IAPP: Flat rate of EUR 1800 per researcher-month managed by the host organisations to contribute for expenses related to the participation of researchers to training activities; expenses related to research costs; execution of the training/partnership project and contribution to the expenses related to the co-ordination between participants.

For EID and IPD:Flat rate of EUR 1200 per researcher-month managed by the host organisation(s) to contribute for expenses related to the participation of eligible researchers to training activities and expenses related to research costs, as well as to contribute to the expenses related to the co-ordination between participants.

Maximum of 10% of the total EU contribution.

10% of direct costs except for subcontractors and the costs of the resources made available by third parties which are not used in the premises of the beneficiary.

EU27 and Associated Countries correction coefficients (adapted from Table 3.2 of the WP)For other countries (such as ICPC and third countries), please consult the WP.

Austria 106.2 France 116.1 Luxembourg 100 Spain 97.7 Albania 63.1 Montenegro 65.0

Belgium 100.0 Germany 94.8 Malta 82.2 Sweden 118.6 Bosnia & Herz. 74.4 Norway 140.6

Bulgaria 62.7 Greece 94.8 Netherlands 104.1 UK 134.4 Croatia 83.0 Serbia 74.0

Cyprus 83.7 Hungary 79.2 Poland 77.1 FYROM 60.6 Switzerland 119.6

Czech Republic 84.2 Ireland 109.1 Portugal 85.0 Iceland 95.0 The Faroes 134.1

Denmark 134.1 Italy 106.6 Romania 69.5 Israel 96.4 Turkey 98.4

Estonia 75.6 Latvia 74.3 Slovak Rep. 80.0 Liechtenstein 109.9

Finland 119.4 Lithuania 72.5 Slovenia 89.6 Moldova 64.3