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Periodic Report Summary 2 - THERAGLIO (Microbubble driven multimodal imaging and theranostics for gliomas)

Project Context and Objectives:
TheraGlio aims at developing combined imaging technologies for diagnostic and tailored therapeutic interventions for patients bearing MGs. This will be carried out by creating a novel multimodal imaging system, which will employ new generation MBs that can simultaneously act as contrast agent for Magnetic Resonance Imaging (MRI), intra-operative Contrast-Enhanced US. Imaging (CEUS) and intra-operative fluorescence-guided microscopic resection of MG. Moreover, newly generated MBs will be loaded with specific targeting molecules and chemotherapeutics for localized release. The specific objectives of the project will be:
1) Designing a new neurosurgical navigation system to simultaneously acquire intra- operative US and operative-microscope images, and match them with the pre-operative MRI scans in real-time.
2) Manufacturing and preclinical assessment of stability, toxicity and efficacy of lab-scale and clinical-grade GMP certified multimodal lipidic MBs as an intra-operative neuronavigation tool.
3) Manufacturing and physical characterization of polymeric (bio-inert or biodegradable), multifunctional MBs, which will be functionalized for MRI, US and fluorescence microscopic visualization.
4) Development of multifunctional drug-loaded-nanoparticle equipped biodegradable MBs as drug delivery platform.
5) Clinical assessment of feasibility, toxicity, and efficacy of multimodal modified lipid stabilized MBs for real-time MR/US image/fluorescence guided surgery using an integrated neuronavigation platform in recurrent GBMs.

Project Results:
WP1 Advanced image-guided neurosurgical navigation platform development
• Development of the new advanced image guided neurosurgical platform which is actually under use by clinical partners P1 FINCB and P3 TASMC (Task 1.7) according to a specific protocol and upon IRB approval. In details:
o Expansion of the algorithm from Task 1.2 so that also other structures (i.e Ventricles) are also considered while doing the tuning
o Use CEUS 3D volumes to extract additional internal landmarks
o Development of a non-rigid registration algorithm where the vessel trees extracted from 3D US volumes are registered.
o Microscope sensor holder design and realization. The holder allows to fix steadily a sensor of the EM tracking system to Microscope, while at the same time allowing to satisfy the requirement of a sterile environment and tools.
o Microscope stereo image calibration, that needs to be performed only once after having placed the sensor holder, and is carried out acquiring with the microscope and appropriately processing a video of a calibration checkerboard.
o Microscope stereo reconstruction, allowing for the 3D reconstruction of the scene viewed through the microscope opticals, and its later fusion together with other 3D data, spatially referenced in the same coordinates system.
o Microscope tracking, by exploiting the same EM tracking system used by the US system.
o Technical integration and data exchange between all the components developed, allowing to gather data on the positions and orientation of the microscope and US probe, and consequently of the data acquired from both imaging systems.
o Ultrasound image, MRI data and microscope 3D reconstructed video integration in a single, VR-like visualization panel, either on a standard monitor or on 3D monitors.
• Preliminary evaluation of the platform in order to collect a feedback about effectiveness, usability, reliability, ergonomicity etc. (still ongoing)

WP2 Production, physicochemical and in vitro characterization of the modified lipid-stabilized commercially available MBs
• Lipid modified microbubbles have been obtained both at lab and quasi-GMP scales.
• Appropriate characterization protocols have been also developed.
• Technology has been transferred to industrial site (Biopraxis), through the intensive collaboration of the WP partners.
WP3 Development and characterization of multifunctional polymer-stabilized MBs
• Indocyanine green (ICG) -labeled PVA-MBs for NIR imaging were synthesized.
• PVA-MB shell was decorated with iron oxide nanoparticles (SPIONs) for MRI imaging.
• Optimization of SPIONs decoration based on TGA results.
• Chemical conjugation of the cyclic peptide (Arg-Gly-Asp-D-Phe-Cys) with PVA shelled MBs (RGD-decorated PVAMBs)
• Biodegradable polymer based (methacrylated derivatives of hyaluronic acid and dextran) vesicles ↔ MBs were synthesized.
• AFM on PVA-MBs: by topography and elasticity analysis the following parameters were assessed:
▪ 4.5 µm sized
▪ shell thickness of 200 nm
▪ surface roughness of 10 nm, with aliphatic chains protruding towards the medium for 10-100 nm
▪ stable Young Modulus of 108 Pa order of magnitude.

WP4 Development and multifunctional nanoparticulated drug delivery system
The research work conducted in this period allowed us to:
• investigate alternative synthetic routes to render NPs Synthesis cheaper, greener, and even more scalable.
• select of a panel of surface functionalisations that are suitable for NP colloidal stability, MB crosslinking, drug loading and at the same time meet criteria for future synthesis scalability

WP5 Assessment of toxicity and safety
Summary of new in vitro cytotoxicity results:

▪ HEK-293 cells show increased cell death supposedly due to apoptotic processes in response to multiples of HED of the RGD-MBs
▪ HUV-EC-C cells show some reaction to multiples of HED of the RGD-MBs (naked MBs were not tested)
▪ SK-N-SH and HEP-G2 cells show not remarkable reactions either to naked or RGD-MBs
▪ SonoVue, SonoVue-Like and naked MBs cause no or only mild reactions in all of the tested cell lines (comparable to non-treated control)
Summary of the in vivo immunotoxicology results:
▪ 100% HED of the RGD-LMB-ICG causes severe CARPA reactions (respiratory and cardiac arrest with skin reaction and long-lasting WBC changes – Gr increase, Ly decrease)
▪ SonoVue and SonoVue-Like cause the same, moderate, tolerable reaction
▪ 100% HED of the naked LMB causes similar reactions to SonoVue / SonoVue-Like
▪ 10% HED of the RGD-LMB causes similar reactions to 100% HED of the naked LMB
▪ Clear dose-dependence exists in the case of both the naked LMB and the RGD-LMB
▪ Partial, weak tachyphylaxis exists in the case of RGD-LMB
▪ ICG alone don’t cause reaction
In brief:
▪ RGD-LMBs in higher doses cause reactions both in in vivo immunotoxicology and in vitro cytotoxicity model systems
▪ Naked LMB-ICGs have mild effects, comparable to reaction evoked by SonoVue / SonoVue-Like
▪ Adverse reactions and cytotoxicity caused by naked or RGD-LMBs are dose-dependent
▪ Kidney cells are the most susceptible to the effects of RGD-LMBs among the tested cell lines; this finding correlates well with some of the conclusions of rat tissue distribution studies
WP6 In vivo functionality of the MBs in rat brain tumour models
In view of the reported findings and under the conditions of this study, it may be concluded that:
• a single intravenous (IV) injection of any of the Test Items Naked Microbubbles (MB), MB-ICG, MB-RGD and MB-ICG-RGD at a dose level of 252 mg/kg is considered to be the no-observed-adverse-effect-level (NOAEL) for each Test Item. The 10-fold higher dose level of 2520 mg/kg of any of the Test Items was associated with adverse effects. Compliance with good laboratory practice.
• following a single intravenous (IV) administrations of the Test Items Microbubbles (MB)-ICG and MB-RGD -ICG to male Sprague-DawleyTM (SDTM) rats, the Test Items are present at the brain. It is likely that the Test Items are also distributed to other tissues, however because of the limitations of the imaging techniques, biodistribution to other organs cannot be proven.
WP7 Clinical Trial Governance
Only preliminary activities related to IRB approval have been performed.

WP8 Performance of a dose escalating clinical trial – this WP will start at month 46

WP9 Dissemination Exploitation
▪ 54 dissemination activities among oral presentations and posters
▪ 6 peer-reviewed publications

Potential Impact:
TheraGlio will contribute to the expected impacts listed in the work programme in relation to topic HEALTH.2013.1.2-1: Development of imaging technologies for therapeutic interventions in rare diseases as follows:
• The development of new and improved technologies for therapeutic interventions in groups or categories of rare diseases
The project is expected to develop and validate novel combined imaging technologies for personalized therapeutic interventions, for a rare form of human cancer, malignant glioma. TheraGlio will likely provide a device for the integration of MR imaging, US imaging, and optical intra-operative visualization, down to the molecular level of malignant brain tumours. It will support the proof of-principle of using MBs as a platform for targeted delivery of therapeutic molecules, which coupled with its properties as medical imaging agent will open an innovative approach for Theranostic (therapy+diagnosis) in MG treatment. This will potentially lead to a significant improvement in defining the tumour extension, understanding the disease biology, determining the functionality of the nearby normal brain tissue, and improving the patients’ overall survival (eventually, the most relevant clinical outcome parameter). In fact MG are not curable because radical tumor resection is not achievable in most of the cases; besides, even when this is feasible, still residual invading cells represent the culprit of recurrence. This is why the TheraGlio project, with its holistic approach, aiming at better visualizing (intraoperatively) and resecting MB, also provides the tool (engineered/modified MBs) to locally deliver an effective chemotherapic drug to those residual cells, expecting to contribute to a longer survival of patients when compared to the current context of extremely poor prognosis. Since a better tool to intraoperatively visualize MG is needed (along with the constant search for new ways of drug delivery to better molecular targets), the impact of the project would go far beyond the expected endpoints of the study. Implementation of project results would possibly improve the outcome of other severe neoplastic conditions where surgical radicality and better drug delivery is important.
• Facilitating the uptake of personalized medicine into clinical practice and support the competitiveness of Europe in this area
It has been shown that MG in general and Glioblastoma in particular represent a vast and heterogeneous pathological entity. In fact, it is well accepted that under the name GBM, we actually group many different tumours, which are characterized by distinct and peculiar genetic mutations that reflect varying level of biological aggressiveness. In this regard, it is clear that not all MB may benefit from the same drugs, since different molecular targets need to be reached in different subgroups of patients.
• The applications are expected to advance research in personalized medicine and have an impact in the relevant industry (in particular for SMEs).
TheraGlio will bring about key possible applications in the field of personalized medicine for the treatment of MG (see above). The achievement of those goals will lead to a most big impact in the relevant industries, particularly in regard to the SMEs that take part to the project. If we will manage to show that the integrated navigation system is effective in showing the lesion pre- and intraoperatively (allowing for larger tumour resection) the project achievements will be particularly relevant for SeroScience, MedCom, ESAOTE, Camelot, and Biopharma. Novel MBs, their related production methods and the local drug-delivery system through NPs, carried by MBs, will be interesting to FINCB, UNIROMA2, TASMC, NANOMOL and BIOPHARMA, as proven technological platforms to be exploded in cancer, degenerative and cardiovascular diseases treatment, consolidating or opening new business lines. Project partners will look for co-development and exploitation with other European SMEs and industries, fostering open innovation schemes.
• The projects will contribute to the International Rare Diseases Research Consortium (IRDiRC).

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