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Microbubble driven multimodal imaging and theranostics for gliomas

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

Executive Summary:
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 US Imaging, including Contrast-Enhanced US (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 multimodal lipidic MBs as an intra-operative neuro-navigation 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 evaluation of feasibility, usability and precision of the multimodal platform using commercially available MBs (SonoVue) in combination with ICG (both EMA-approved) for real-time MR/US image/fluorescence guided surgery using an integrated neuronavigation platform in recurrent GBMs; proof of principle test to validate the functionality of the new platform in vivo.

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 US Imaging, including Contrast-Enhanced US (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 multimodal lipidic MBs as an intra-operative neuro-navigation 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 evaluation of feasibility, usability and precision of the multimodal platform using commercially available MBs (SonoVue) in combination with ICG (both EMA-approved) for real-time MR/US image/fluorescence guided surgery using an integrated neuronavigation platform in recurrent GBMs; proof of principle test to validate the functionality of the new platform in vivo.

Project Results:
WP1 Advanced image-guided neurosurgical navigation platform development

The WP1 was focused on the development of an innovative image guided neurosurgical platform aimed at combining pre-operative MR and CT scans with intraoperative US and microscopy images into a common 3D interactive scenario. The goal of the platform is to support the surgeon before and during glioblastoma removal in order to support the maximization of tumor resection, the prevention of post-operative neurologic deficit and the identification of deeper structures of interest in the trajectory of resection. The platform combines functionalities for both therapy planning and intraoperative guidance, as well as post-operative evaluation.
Concerning therapy planning functionalities significant effort has been dedicated to developing a system that allows the surgeon to accurately plan the neurosurgical entrance direction and brain the operation tunnel on a 3D reconstruction of the patient’s skull and brain. Towards this aim, algorithms for accurate pre-operative multimodal neuro-image fusion, re-slicing of all modalities, 3D skull visualization from CT have been implemented. Further functionalities that have been integrated in the platform are related to planning and positioning of head holder for neurosurgery and automatic head key-point detection and visualization. Therefore, a new neuro-navigation workflow, which switches layouts and enables features automatically, thus minimizing the input needed from the doctor has been designed, In addition has been added a registration environment for displaying and setting the external markers directly in the acquired volume slices or on a reconstructed 3D volume, allowing very fast registrations within less than a minute
Furthermore an algorithm which rotates the reconstructed 3D head face-up, thus matching the real-world situation and providing better guidance and orientation for the physician has been implemented.
Concerning intraoperative guidance, to enable effective intra-operative image fusion, the integration of the ESAOTE MyLab Twice Ultrasound system and tools with the neurosurgical ZEISS Pentero 900 Microscope used during the surgery has been addressed. This integration is aimed at combining the intraoperative optical imaging from the operating microscope with the ultrasound imaging and the available pre-operative images. In order to integrate all the available information in a common virtual environment, the microscope tracking, the stereo video input management, the integration with the ESAOTE MyLab Twice ultrasound system and the generation of an augmented virtuality composed by all of the acquired data have been addressed. In particular a 3D representation of the scene viewed through the operating microscope is obtained from the stereo images captured by the surgical microscope exploiting different computer vision algorithms. Microscope position and orientation tracking has been obtained thanks to the integrated electromagnetic tracking tool available in the employed US machine, and a dedicated calibration procedure has been developed to properly integrate the obtained reconstruction with the pre-operative MR/CT scans and the intra-operative US into the same virtual scenario.
Several experimental sessions have been performed in order to evaluate and validate the overall system integration, using both artificial and ex-vivo phantoms, exploited to evaluate different aspects of the developed procedure. Artificial phantoms were exploited to both qualitatively and quantitatively evaluating the accuracy of the major steps of the developed image fusion pipeline, including the reconstruction of the microscope field of view from the stereo images, the automatic localization of the reconstructed brain surface in the reference system defined by the US system, and its integration with the other available data. To quantitatively assess the accuracy of the developed reconstruction pipeline, the 3D reconstruction of the phantom’s surface obtained from the adopted stereo-reconstruction algorithms has been compared to the one obtained using a portable multi-pose 3D scanner based on infrared depth-sensing technology, with satisfactory results. As regards the calibration and localization process, the positioning of the obtained reconstruction of the phantom surface in the reference frame provided by the electromagnetic (EM) transmitter integrated in the US machine has been evaluated by measuring the surface distance to the available MRI surface reconstruction. In this phase, the adoption of an artificial phantom with known landmarks allowed an accurate registration between MR scans and the EM frame, leading to an optimal integration of data, and avoided the registration errors caused by the brain-shift effect allowing the evaluation of the errors solely introduced by the surface’s reconstruction and localization in the reference system.
The subsequent adoption of an ex-vivo bovine phantom was aimed at evaluating the fusion results achievable considering a set of data similar to real patient’s data. In these cases, a qualitative analysis on the efficacy of data integration has been carried out, initially by evaluating of the quality of the brain’s reconstruction and localization, and later by investigating to what extent a joint data visualization could enhance the available information. Finally usability of the overall system was also evaluated and optimized.



WP2 Production, physicochemical and in vitro characterization of the modified lipid-stabilized commercially available MBs

The activities carried out during WP2 were focused on the production and modification of the prototype of lipid microbubbles (MBs) to enable their visualization in three different modes: US, MRI and intra-operative fluorescence microscopy. Commercially-available lipid MBs SonoVueTM (Bracco), MBs already approved by EMA for use in humans and routinely used by the partner P1 FINCB intraoperatively for malignant gliomas (MG) resection, were used to include those modifications so that the new modified lipid MBs could be more easily translated into the clinical area.
To that end, following the synthesis of SonoVue-derived lipid MBs using sulfur hexafluoride (SF6) or perfluoropentane (PFP), their characterization in terms of size distribution and concentration, labelling of MBs with a fluorescent phospholipid, and the in vitro ultrasound transmission attenuation profile was carried out at laboratory scale at UNIROMA. For that purpose, the SonoVue-derived MBs were first modified by including the near-infrared (NIR) dye Indocyanine green (ICG) in the lipid shell. ICG dye is FDA/EMA-approved for use in humans and due to its high level of absorption in the NIR region, it can be tracked within deeper tissues during the operation. Obtained SonoVue-derived MBs were similar to SonoVueTM, as confirmed by differential scanning calorimetry thermograms. ICG-labelled SonoVue derived MB were successfully developed as observed by optical microscopy.
Secondly, decoration of the MB lipid shell with cyclic arginine-glycine-asparagine, c-RGD, peptide motif (a ligand for integrins αvβ3, αvβ5, overexpressed in the glioma vasculature) in order to modulate the adhesion properties towards endothelial cells was performed. For that purpose, attachment of c-RGD-Cys was carried out to the distal end of the poly(ethylene glycol) (PEG) moieties on the MB surface. Peptide conjugation was performed to maleimide-bearing MBs via thioether bonding. This strategy was preferred to the streptavidine-biotin coupling because it is known the immunogenetic response that can be triggered by these compounds. In fact, if the use of streptavidin is, on one hand, advantageous due to the commercial availability of a number of biotinylated bioactive ligands, which allow the MBs functionalisation without the need to design new MBs for each vascular molecular target under investigation; on the other hand, repeated injections of streptavidin-carrying particles can determine the risk of an immune response. Therefore, streptavidin-containing MBs are unlikely to be clinically translated. The functional in vitro adhesion of the developed optimised MBs was demonstrated in endothelial cells (HUVECs).
Once the optimised SonoVue-derived MBs were developed at laboratory scale (UNIROMA), the production process was transferred to GMP pilot scale (BIOPRAXIS). To that end, the process was optimised in several aspects to make it easier scalable and to improve critical atributes such as particle size, concentration and stability. For the preparation of lipidic MBs three different methods were carried out in order to obtained the required batches: liophilisation and compressed CO2-based methodologies, such as DELOS and PCA (NANOMOL). Compressed fluid based DELOS process yielded very low concentrated MB samples, which presented a bigger size than those coming from lyophilisation. Moreover, at the tested operating conditions, which oblige to use a suspension instead of a solution of lipids, DELOS process did not constitute a good alternative to lyophilisation for the preparation of lipidic microbubbles. On the other hand, compressed fluid PCA process provided MB samples which presented similar size characteristics than those prepared by reconstituting a lyophilized cake. However, the concentration of MBs in the sample was lower for the compressed CO2-based methodology, which needed optimisation in order to constitute a good alternative to lyophilisation for the production of lipidic MBs.
Finally, liophilisation was chosen as the method of choice to produce optimised lipid MBs. In order to scale up the production process under GMP conditions, a considerable amount of pilot batches were produced for their characterization in order to get the optimum conditions. The scaled production process was designed, including sterility needs, areas, equipment, assessment of critical process variables, Standard Operational Procedures drafting and a GMP production program was set up by BIOPRAXIS. A great ammount of work was carried out in order to optimise the process, such as filtration, freezing, freeze-drying, SF6 incorporation and esterilisation optimisation.
On the other hand, an extensive characterisation work was carried out (NANOMOL) with the batches produced at both, laboratory and GMP scale. The techniques used for that purpose were laser diffraction (Mastersizer 3000), an ensemble particle sizing technique that delivers volume-based particle size distributions.This technique is suitable for many applications within the pharmaceutical sector as it is particularly sensitive for the detection of over-sized material, and it is well-established as a process analytical. Automated optical microscopy (Morphologi GS3) was also used for characterisation, as it allows to automatically analyse the size and shape of thousands of particles in a short period of time. Apart from providing a validation of particle size distribution results coming from laser diffraction, this equipment also allows to make a full morphological characterization of particles and to obtain values of concentration of MBs once reconstituted with the same buffer used for administration to patients.
While optimising the production process under GMP, the toxicological studies carried out within WP5 revealed that cRGD-MB shown a high CARPA reaction in pigs compared to marketed Sonovue. The high toxicity shown by the developed cRGD-MB were unacceptable to carry out a clinical trial in humans; thus, the THERAGLIO Consortium decided to cancel the final production of GMP grade pilot batches of cRGD-MB.


WP3 Development and characterization of multifunctional polymer-stabilized MBs

This Work package is focused to the development and characterization of multifunctional polymer shelled microbubbles (MBs).
The activity was mainly addressed to the synthesis, engineering and characterization of (i) poly(vinyl alcohol) shelled MBs and of (ii) bioinert and biodegradable biopolymer (dextran or hyaluronate) shelled MBs.
Within Task 3.1 MBs of type (i) were modified for US/fluorescent/MRI imaging, cell targeting and drug delivery without jeopardize the starting good acoustic performances of the unmodified poly(vinyl alcohol) shelled MBs (PVA-MBs).
As for fluorescent and magnetic resonance (MRI) imaging PVA-MBs were functionalize with indocyanine green (ICG) fluorophore for near infrared (NIR) and with super paramagnetic iron oxide nanoparticles (SPIONs), respectively.

RESULTS
Labeling PVA-MBs with ICG
ICG was chosen as already validated by NIH for clinical use and in particular in neurosurgery intervention. ICG was covalently linked to the PVA-MB surface using the Sulfo-NHS (N-hydroxysulfosuccinimide) derivative of ICG which can be reacted with hydroxyl groups present on the PVA shell of MBs. The structure of the ICG dye used, ICG-PEG8-Sulfo-OSu (Dojindo), is shown in Fig.1; the short PEG arm increases the hydrophilicity of the molecule.

The confocal microscope of the unit UNIROMA 2 was implemented with an ad hoc optical filter in order to test the efficacy of this dye in the NIR field.
The “red rings” shown in Figure 2 correspond to the equatorial planes of ICG labeled PVAMBs with size of about 3 μm.

PVA-MBs for MRI
SPIONs were obtained from MagForce (Germany). The attachment of SPIONs to PVA-MBs was physical, including of SPIONs in the PVA shell of MBs during their formation, or chemical, anchoring the SPIONs via chemical link to the surface of MBs.
Table 1 summarizes the content of iron in the PVA-MBs obtained by the two methods.


The results reported in Table 1 clearly show that the magnetic iron nanoparticles chemical tethering is much more efficient.
MBs were tested with an external magnet to test the responsivity of PVA-MBs functionalized for MRI.
Task 3.2 of WP3 concerned type (ii) polymer based MBs enabling multimodal imaging, cell targeting and drug loading”
The synthetic strategy used to obtain type (ii) MBs was in three steps (see Scheme 1):
A) deposition of a surfactant layer around a perfluorocarbon (PFC) droplet;
B) the deposition, as the outmost layer on the water/droplet interface, of an amphiphilic polymer grafted with a vinyl moiety
C) UV curing to crosslink the grafted vinyl side-chains via radical polymerization.
These droplets, irradiated by ultrasound (US), undergo an acoustic droplet vaporization process, ADV, which allows liquid ↔ gas transition in the core, transforming the polymer shelled droplet into MBs with a reversible change of particle dimensions. This process is readily observed by the floating tendency of the MBs and can be monitored using laser scanning confocal microscopy (CLSM), labelling the polymer shell with rhodamine B isothiocyanate, RBITC. In CLSM images the droplets/microbubbles are visualized as rings after focusing the equatorial plane of fluorescent labelled microparticles (Fig. 4).
The surface of PVA-MBs were functionalized with the cyclic peptide, cyclo Arg-Gly-Asp-D-Phe-cys), c-RGD-Cys, shown in Figure 5. In Nature the RGD sequence is an essential recognition site for cells expressing αvβ3 integrins [Annu Rev Cell Dev Biol 1996;12:697–715]. The RGD ligand molecule has been anchored to the surface of PVA MBs via maleimide-thiol conjugation as summarized in Scheme 2.
The adhesion efficiency of the RGD decoration was tested with the in-house designed set up shown in Scheme 3.
In a typical experiment a suspension of PVA-MBs decorated with RGD were fluxed at physiological shear stress in microchannels where HUVEC cells were attached, thus mimicking the physiological conditions. PVA-MBs/RGD where significantly more efficient in adhere to HUVEC cells than the control (PVA-MBs without RGD). A study on the dependence of the efficacy of adhesion from extent of RGD surface coverage and shear stress in important to understand the mechanism of adhesion of theranostic devices to cells.
Other adhesion agents were also considered, such as αVEGFR2 and Transferrin. However, RGD was evaluated from an ad hoc study the most efficient in assuring the tethering of MBs to HUVEC cells, as reported in Figure 7.
Elasticity and morphology of MBs surfaces was accomplished by Atomic Force Microscopy, AFM (Task 3.4).
AFM Profile of intact and bursted microbubbles is reported in Fig 7



WP4 Development and multifunctional nanoparticulated drug delivery system

The Objectives of this work package was the design of targeted drug-loaded NPs to be bound to MBs as multifunctional contrast agent for multimodal imaging and as a drug delivery platform for MG cell directed therapy. The delivery by immobilized MBs will overcome some intrinsic problems of targeted, nanoparticulated drug delivery systems, namely the high dilution of NPs in blood after i.v. injection and the short residence time of NPs in close vicinity to the vessel wall to extravasate.
During the project several magnetic iron oxide or iron oxide-gold alloy nanoparticles were prepared exposing different functional groups on the surface in order to allow binding to the microbubble surface and here in particular to PVA. Finally, the commercially available and EMA approved magnetic iron oxide nanoparticles, NanoTherm® from MagForce were used as MRI (magnetic resonance imaging) visible core. These nanoparticles were coated by a polymer in order to immobilize and protect the temozolomide which intrinsically is instable in hydrophilic environment such as plasma as well as increase the loading with the hydrophobic drug. The polymeric shell was covered with perillyl alcohol for targeting and improved efficiency of temozolomide as it was reported in literature for gliomas or metastatic brain cancers (e.g. doi: 10.1093/neuonc/nou208.48). Albumin was used on the outer surface to stabilize the nanoparticles. In previous experiments we could show that albumin coating of nanoparticles enhance the passage through the blood brain barrier (Sousa et al., 2010; Schäffler et al., 2014). The nanoparticles were characterized and tested for cell toxicity in different cell line such as mouse brain endothelial cells or glioma cell lines. The particles showed a low level of toxicity to the endothelial cells while the toxicity of the released temozolomide is comparable to that of free temozolomide for the tested glioma cells.
In order to allow a targeting for the tumor different from antibody targeting or the binding to integrins expressed on the vessel surface of tumor microvasculature we explored different lectin libraries on brain tumor samples from patients. As part of the work also a protocol to bind the lectins to nanoparticles were developed. While some lectins showed a good affinity and staining of vessels or tumor cells it became clear that only a mix of lectins can allow to properly distinguish the tumor tissue from normal one. This work will lead to future studies in which we will test the selectivity of multi-lectin presenting nanoparticles to glioma cells either invading in the normal tissue or on the boundaries of the surgically removed primary tumor.




WP5 Assessment of toxicity and safety
Complex nano,-micro-constructs which can fulfill the requirements of today’s medicine may integrate multiple functions like targeting, prolonged circulatory time, diminished toxicity, can serve as therapeutic and diagnostic tools in a combined multifunctional theranostic. Besides their advantageous properties these structures often provoke unexpected and sometimes severe immunotoxic reactions. The immune system recognizes the i.v. injected complex drugs as foreign particles and initiates an acute defense reaction with an atypical, non-IgE-mediated pseudoallergic reaction. To characterize, predict, and prevent pseudoallergic reactions to nanomedicines, which often arise following their first intravenous administration, EMA recommends the detection of the Complement Activation Related PseudoAllergy (CARPA).
SeroScience Ltd. – besides the in vitro tests developed a sensitive in vivo pig model to detect the possible - otherwise unpredictable - acute cardiopulmonary distress, which can be severe or occasionally lethal, and therefore, unacceptable for a theranostic. It should be emphasized that the swine model does not represent the reactivity of the average human population, but the most sensitive individuals who show mild or severe (rarely fatal) reactions after nanomaterial injection / infusion - that is, 2-7% of the population.
To develop such complex structures needs very careful and tedious work until the development from bench to the first clinical trial, to bedside. A further complication is that any structural or technological change, any addition to the complex structure may influence the reactogeneicity – and this necessitates the immunotoxicity testing during each development step, not only testing the final product.

Summary of toxicity and safety testings during the project:

Period 1
▪ Obtainment of the ethical permission for the animal experiments; preparation for the in vitro
citotoxicity works; in vitro study of the first (naked) lipidic and PVA microbubbles; in vivo testing of
the above compounds
▪ PVA (polyvinil alcol) and lipidic microbubbles (LMBs) were tested in vitro in 3 human sera, and
also in pig CARPA model (2 pigs). With both methods both compounds were reactive, caused
complement activation (sC5b-9 increase) and pseudo-allergy reaction – CARPA in pigs. The reaction
was moderate, caused pulmonary pressure elevation, was dose dependent and did not show
tachyphylaxis.

Period 2
Summary of 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
Period 3
Summary of in vitro testing of MBs as well as different targeting molecules regarding in vitro immunogenicity in human sera:
In vitro sC5b-9 tests in human sera were conducted in two series:
▪ PVA-MBs with different amount of MagForce spions were decorated and applied repeatedly in the tests.
- 4x2 different PVA-MBs + MF with 2 different concentration (final conc.: 2,5E+08 and 2,5E+07 MBs/mL) and MagForce (final conc.: 1, 10 and 100 µg/ml) were tested in 3 different human sera.
- MagForce spions alone did not cause complement activation in any applied dose.

▪ Testing of possible targeting molecules regarding immunotoxicity in 3 doses in human sera:
- Four different targeting molecules (Cell adhesion mol.; Holo-Transferrin; Lactoferrin; Anti-KDR) with 3 different concentrations were tested in 3 different human sera by sC5b-9 EIA kit. There was no significant complement activation after any molecules in any applied dose (max. changes: 0,55-2,7x of PBS) and he results did not show dose dependency.
In vivo testing of immunotoxicity of lipid MBs in big animal pig model:
▪ RGD-LMB-ICG test materials were administered in different order to the 4 pigs involved in the experiments.
- 5-10-20% RGD coverage of the LMBs with ICG cause moderate CARPA reactions (with PAP, SAP and mild, transient WBC changes)
- SonoVue causes mild, tolerable reaction
- There is no significant difference between 5-10-20% coverage of the RGD-LMBs regarding immunotoxicity
- Partial, weak tachyphylaxis were detected after RGD-LMB-ICGs application
Last reporting period
In vivo testing of immunotoxicity of PVAMBs and multimodal-PVAMBs in big animal pig model:
▪ In vivo immunotoxicity of the final GLP produced poly(vinyl alcohol) shelled microbubbles, PVAMBs, and multimodal microbubbles supporting magnetic resonance imaging, fluorescence and ultrasound imaging with targeting capability, i.e. microbubbles with a surface containing magnetic nanoparticles MagForce(MF)-fluoresceine isothiocianate(FITC)-cycloRGD-PVA microbubbles, called “multimodal-PVAMBs” were tested in 7 domestic pigs.
- The new generation, multimodal microbubbles (MBs) that can simultaneously act as a contrast agent for Magnetic Resonance Imaging (MRI), intra-operative Contrast-Enhanced US Imaging (CEUS) and intra-operative fluorescence-guided microscopic resection of MGs did not show severe immunotoxic symptoms.
- None of the analyzed immunotoxicity parameters showed statistically significant difference between the marketed reference material SonoVue and the two test materials, the PVA–MBs and MF-PVA-MBs.
- It is a great (and in the SeroScience’s practice rare) achievement that a complex, multimodal theranostic after a long development reached the aim and cause only acceptable immunotoxicity reactions in the sensitive pig model.
In vitro testing cell toxicity of PVAMBs and multimodal-PVAMBs in cell culture models:
The experiments are ongoing and the Report will be delivered at the end of this week.





WP6 In vivo functionality of the MBs in rat brain tumour models

Period 2
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.
Period 3
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 Study Governance

A new study protocol was defined according to the Amendment procedure. The study was submitted to FINCB IRB and approval was obtained on July 2017.
Title: THERAGLIO - EVALUATION OF A NEW INTEGRATED PLATFORM FOR PRE- AND INTRAOPERATIVE IMAGING IN GLIOMA SURGERY (observational prospective cohort study)


WP8 Performance of the clinical study to evaluate the new integrated neuro-navigation platform

The innovative image guided neurosurgical platform developed within the WP1 was employed for clinical evaluations within the scope of WP8. The clinical evaluation main aim was the assessment of the platform’s performances on patient’s data, with particular focus on the precision and usability of the developed algorithms and tools. Several experimental sessions have been performed to acquire and elaborate patient’s data in order evaluate the platform’s ability to reconstruct the brain’s surface from the microscopic stereo images and to correctly integrate it with intraoperative US, and other available data (e.g. pre-operative CT, MR) in real case scenarios.
Since from initial evaluations it emerged that the procedure needed to properly calibrate the microscope and localize the reconstructed surface in the virtual scenario might have extended the total duration of the surgical intervention, the localization process has been preliminarily evaluated and optimized on artificial phantoms in order to minimize the patient’s risk. As, the accuracy of the localizing procedure does not depend on the type of reconstructed object, the results achieved during this experimental session have been considered representative of the overall accuracy achievable in a surgical environment.
Several further experimental sessions have been performed on patients undergoing glioblastoma removal to assess whether a combined data visualization may support the surgeon during the intervention. All these activities were conducted in the contest of the clinical study, although not all the objectives were achieved, the most fundamental landmarks were reached in order to ascertain the platform utility and sustainability in a clinical setting. In this regard, we had to face some unexpected issues like the study protocol amendment procedure submission and approval along with some technical matters related to a more complex surgical flow where the implementation of the platform prototype has made surgical procedure more prolonged and cumbersome.
Multimodal data have been recorded before, during and after surgery to evaluate the platform’s capabilities at different stages of the surgical procedure. During these experiments, a preliminary analysis on the stereo reconstructions’ quality has been performed in order to optimize the effectiveness of the brain’s surface representation. The stereo images representing the surgeon field of view have been acquired using a surgical microscope at different timepoints during the surgical intervention (e.g. prior, during and after the dura mater’s opening and the tumor’s removal). Since the brain’s surface is characterized by prominent features (such as vessels, bleedings etc.), a broad set of point correspondences could be identified in the stereo images, allowing for a final satisfactory surface representation. The integration of the pre-operative CT and MR images with the intraoperative US volumes and the microscope 3D reconstruction of the brain’s surface enabled a comprehensive final representation of data, allowing interactive navigation of all the available modalities in a virtual 3D environment. Based on the results of the usability evaluation it can be claimed that the possibility to analyze the collected data from several viewpoints in the virtual scenario, together with the possibility to select different direction of data visualization and to display the data from the surgeon point of view can facilitate and speed-up the evaluation of the structures of interest.


WP9 Dissemination Exploitation

A widespread reaching number of dissemination activities has been achieved by the Consortium, at local, national and international levels, in order to promote the visibility of TheraGlio to the scientific and industrial community.
During the kick-off meeting of TheraGlio in Milan on October 28-29, 2013, a dissemination plan was designed for identifying the targeted audiences developing the best communication objectives, the activities and tools to be carried out, with details on the rationale behind the selected strategy, as well as the timing and responsibilities for this implementation.
The partners decided to implement two different levels of dissemination: one was aimed to raise awareness on the project existence, its objectives and previewed impact, while the other one was aimed to spread more technical information, based on project results. The first level of dissemination addressed a non-scientific audience, such as the patients’s associations and the general public, including the press, where relevant, while the second level was mainly used to inform the scientific and industrial community about the project achievements.
For maximum visibility, a clear and precise identity, linked with a graphically coherent and consistent representation was created.
The dissemination toolkit consisted of the project logo, the project website and a project leaflet / brochure.
The logo represents a brain from which bubbles of different sizes emerge. The image is meant to emphasize the focus of the project novel technology, which is based on the use of microbubbles in imaging both in the diagnostic and in the surgery phases of the brain.
The project website (www.theraglio.eu) was created and maintained by CAMELOT, and regularly updated throughout the project duration. The structure and layout contains key and easily accessible information about the project, its objectives, and organization, consortium partners, news and events, dissemination activities and results such as publications. The website acknowledges FP7 support and incorporates the EU flag and European Union Seventh Framework Programme (FP7) logo.
A press release was issued at the beginning of the project to announce the awarding of the Grant by the European Union Seventh Framework Programme to Fondazione IRCCS Istituto Neurologico “Carlo Besta”.
Dissemination of scientific results from the project was done mainly through the submission of articles for publication in high impact peer reviewed journals or in the proceedings of the national/international scientific meetings. To date more than 20 scientific articles were published acknowledging TheraGlio
Moreover, the TheraGlio results have been showcased during 124 national and international meetings by the consortium members.

WP10 MANAGEMENT
The TheraGlio project was based on a coordination structure and decision making mechanisms set up to cope with the complexity of the project. The consortium placed particular attention in designing its management structure in order to have strategies for keeping control on the activities and on the potential problems that may arise.
P1 FINCB has acted as Project Coordinator (PC) and was responsible for the contacts between the EC and the Consortium and responsible for the scientific, legal, contractual, ethical, financial and administrative management in line with Art. II.2.3 of the GA.
P10 CFc supported P1 FINCB in these activities, by collecting and gathering data for deliverable preparation, by providing partners with deliverable templates, and reminding the concerned partners about deliverables deadlines, and by editing documents to be submitted to the Coordinator.
In order to ensure an effective and collaborative environment, P1 FINCB, with the support of P10 CFc, took care of setting up and maintaining appropriate mailing lists according to the needs of the consortium. Moreover, P1 FINCB, with the support of P10 CFc, organized annual/periodic face-to-face project meetings where project- or consortium- wide technical and managerial issues were addressed and where partners were able to collaborate in the review of the project’s progress and against original (and in light of future) planning.
Strategic planning and project driving was responsibility of an Executive Committee (ExC),consisting of the Project Coordinator and one representative from each partner.
The ExC was in charge to supervise the overall direction of the research project and monitoring the progress.
The TheraGlio consortium organized a kick off meeting at the start of the project and five annual meetings.
Along the project duration, WP 10 activities were also related to the overall financial and administrative management, including the management of the EC contribution, the distribution of the EC payments, the monitoring of the schedule (including the achievement of deliverables and milestones), and the set up of a framework for the preparation and submission for the periodic reports.
The project has faced three amendments to the Grant Agreement, due to changes to the consortium, to revisions due to technical issues.

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:
• Impact on scientific production (publications)
Thanks to the THERAGLIO scientific activities, more than 20 scientific articles were published acknowledging TheraGlio. Moreover, the TheraGlio results have been showcased during 124 national and international meetings by the consortium members.

• The development of new and improved technologies for therapeutic interventions in groups or categories of rare diseases
The project has set the basis for the development of novel combined imaging technologies for personalized therapeutic interventions, for a rare form of human cancer, malignant glioma. Preliminary studies on phantoms and in vivo have been performed, which will eventually lead to the implementation of a device for the integration of MR imaging, US imaging, and optical intra-operative visualization. It has supported 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 MG, also explored the possibility (by means of 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 MG 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 suggested possible applications in the field of personalized medicine for the treatment of MG (see above). The further implementation of this approach will lead to a big impact in the relevant industries, particularly in regard to the SMEs that take part to the project. Showing that the integrated navigation system is effective in showing the lesion pre- and intraoperatively (allowing for larger tumour resection) is particularly relevant for SeroScience, MedCom, ESAOTE, Camelot, and Biopraxis. 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 Biopraxis, 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.

• Scaling up and transferring GMP-like conditions:
The work carried out during the THERAGLIO project has allowed an important scientific impact by demonstrating the ability to achieve multimodal MBs able to better visualise the tumour borders by three different imaging techniques such as US, MRI and intra-operative fluorescence microscopy. This is of great importance for patients bearing malignant glioma as an appropiate tumour resection has been related with higher successful rates. Regarding the technological view, production of optimised lipidic MBs were transferred and succesfully achieved from a laboratory scale to GMP-like conditions. Additionally, methodologies for the characterisation of MB samples coming from development and scale-up activities have been developed and implemented following GMP requirements. This activity has enabled to qualify the equipments and labs working in project for the characterisation of MBs as particulate matter.

• Improving intraoperative imaging and tumor visualization:
The innovative image guided neurosurgical platform developed in the scope of WP1 and WP8 proved to be able to allow the surgeon to explore the available pre-operative and intra-operative multimodal data in an interactive augmented virtuality scenario. This may help surgeons in achieving a more complete understanding of the imaging data during surgery, thus eventually resulting in improved patient outcome and reduced operatory room usage. In particular, the integration of intraoperative US with the microscope 3D reconstruction of the brain’s surface provides information about the structures of interest which can be updated during surgery, thus overcoming the typical difficulties involved in relying on pre-operative data affected by brain shift after dura-mater opening. In future works, both the pre-operative and intra-operative images could be directly injected and fused in the microscope oculars, thanks to their known position with respect to the microscope, thus eliminating the surgeon’s need to consult an external platform, further improving usability and effectiveness of the platform.

List of Websites:
www.theraglio.eu

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