Etape

3 luni

Computer-controlled RFA and MNPs ablation

0 EUR
Obiective
4.1 Mathematical modelling of the (electro)magnetic field generated: UCV 4.2 Finalising control software development: UCV + UMFCV + UMFCN 4.3 Dissemination and website of the project: All Partners – UMFCV + UMFCN + UCV + INCDFM + ICMPP
Activități
Objective 4.1 Mathematical modelling of the (electro)magnetic field generated Task 4.1.1 Computational simulation and testing of a combined RFA/MNP nano-ablation procedure. After our innovative needle design, the properties of the lesions produced in different tissue types for various RFA/MNP regimes will be determined by extensive in-vitro testing. Using tests results and finite elements modeling (FEM) to simulate the cellular heating, impedance variation, thermal exchanges within the tissues, vascularization influence, the shape of the lesions can be predictable in our opinion and will be assimilated with regular 3D volumes (as spheroids for example), with size depending the time the tissues are exposed to heat as will be determined from computation. The proximity of large vessels cools the surrounding area and deforms the shape of the lesion and will be considered during simulation. Objective 4.2 Finalising control software development Task 4.2.1 The algorithm developed during this project will be used for minimization the downhill simplex method because it is the most efficient, combining at best fast computation with accuracy. In order to find efficiently the optimal trajectory, the user will have the option to try various initial positions from the candidate zones proposed by the algorithm as possible to insert a needle. This task will be performed by CO + P1 + P2, based on the clinical experience of UMFCN in the field of conventional ablation. Objective 4.3 Dissemination and website of the project Task 4.3.1 The project website will be structured to reflect the various sub-components, and wherever possible visual clues will be deployed to explain our research to the public-at-large (lay persons). Scientists will be able to download publications and patent applications as they become available. The website will also contain the progress reports, per project, and will also grant access to our team members to an internal WIKI or BASECAMP, where alternatives and critiques of on-going research will be enabled. Project results, avenues for future research protocols as well as the phase 1 and 2 trials of the newly developed MNPs will be described on the website. All partners (CO + P1 to P4) will participate jointly in this task, with dedicated funding being mobilized for the IT component.

12 luni

Combined targeted MNPs and RFA tumor ablation

0 EUR
Obiective
3.1 Preparation of targeted MNPs loaded with anti-angiogenic drugs: ICMPP 3.2 Combined use of RFA and MNPs in normal pig liver and pancreas: UMFCV 3.3 Combined use of RFA and MNPs in human HCC and PAC (tumor explants): UMFCV + UMFCN + INCDFM 3.4 Assessment of the nano-ablation effects as compared to conventional RFA: UMFCV + UMFCN + INCDFM 3.5 Construction of the computer-controlled hybrid needle for US-guided or EUSguided procedures: UCV + UMFCV + UMFCN Description of work (possibily broken down into tasks) and role
Activități
Objective 3.1 Preparation of targeted MNPs loaded with anti-angiogenic drugs Task 3.1.1 Synthesis of MNPs loaded with angiogenic drugs (e.g. sorefenib or bevacizumab) with well controlled conditions to obtain sub-micron dimensions. The drug will be kept on the MNPs surface by covalent bonds or by physical interactions. Thus, MNPs will reduce the exposure of healthy tissues by limiting the distribution to target tumors of interest. This task will be performed by P3 + P4. Objective 3.2 Combined use of RFA and MNPs in normal pig liver and pancreas Task 3.2.1 Intratumoral diffusion of the previously synthetised and tested MNPs will be assessed in normal pig liver and pancreas. MNPs activation will be triggered through an external magnetic field, while the effects will be followed by the size of the ablated zone during follow-up and longitudinal monitoring of tumor angiogenesis changes by CEUS. After 2 to 4 weeks the pigs will be euthanised and the size of ablation will be assessed by pathology. Task 3.2.2 MNPs loaded with antiangiogenic drugs will be also tested, with a similar methodology in normal pig liver and pancreas. Again, the size of the ablated zone during follow-up and longitudinal monitoring of tumor angiogenesis changes by CEUS and targeted US, as well as pathology assessment after euthanasia. This task will be performed by CO + P1. Objective 3.3 Combined use of RFA and MNPs in human HCC and PAC tissue Task 3.3.1 These will be performed on explanted HCC and PAC tumors by using nanoablation, i.e. injection of MNPs with passive diffusion inside the tumors, followed by external activation with a magnetic field. This task will be performed by CO + P1 + P3. Objective 3.4 Assessment of the nano-ablation effects as compared to conventional RFA Task 3.4.1 A comparative pathology study will be performed after conventional RFA in pig liver and pancreas, as well as explanted HCC and PAC tumors, in comparison with nanoablation using newly developed MNPs. In addition, we will perform histological descriptions of the apoptosis / necrosis areas and the “heatsink” effects that follow RFA or nano-ablation. Objective 3.5 Construction of the computer-controlled nano-ablation needle for USguided or EUS-guided procedures Task 3.5.1 The new design of the hybrid RFA/MNP nano-ablation needle will be performed by P2 , based on inputs from CO + P1 and will include: - a computer-controlled actuation of the electrodes using a Maxon stepper motor with gearbox and digital positioning controller placed inside the handler. Through a simple bevel/rack gear mechanism this will adjust the insertion and the deployment angle of the electrodes - a micro-coil placed on the needle central stylet will generate an alternant magnetic field heating the MNPs previously injected in the tumors - the needle will have 3 electrodes from nitinol (material with memory) that will be formed for an optimal shape determined from finite elements simulation. This shape will allow gradual and controllable insertion/deployment to optimize thermal exchange inside tumour Task 3.5.2 The algorithm and software development for optimal needle control during the nano-ablation procedure will be initiated, performed by P2. The algorithm will assess the optimal position for the needle insertion after the user-defined initial position. The main criterion for this optimization will be the volume of the resulting lesion, e.g. to determine the minimum of a function that associates to each candidate needle position the volume of damaged healthy tissue, while still covering the whole tumor. In order to eliminate these trajectories that would damage viable healthy tissue, the algorithm will include also a penalty volume.

12 luni

Assessment of MNP activation and passive intralesional delivery

0 EUR
Obiective
2.1 Synthesis and structural analysis of hydrophilic MNPs: ICMPP 2.2 Functional characterisation of hydrophilic MNPs: ICMPP + INCDM 2.3 Magnetic activation of NPs and passive intralesional delivery: INCDFM + UMFCV 2.4 Assessment of therapeutic effects (apoptosis / necrosis): UMFCV + UMFCN 2.5 MNP validation using the nano-ablation tester: INCDFM + UCV
Activități
Objective 2.1 Synthesis and structural analysis of hydrophilic MNPs Task 2.1.1 Synthesis of magnetite with amino or carboxyl functional groups. Hydrophilic magnetite particles will be prepared by two different methods (co-precipitation and free-solvent synthesis). This task will be accomplished by P3 + P4, based on previous experience. Task 2.1.2 Structural analysis of magnetite particles and MNPs with -NH2 or -COOH groups will be also performed by P3 + P4, based on previous experience. Task 2.1.3 Synthesis of iron oxide nanoparticles by (i) solvothermal method; (ii) ball milling. Task 2.1.4 Exploring novel magnetic materials, such as Fe16N2 with a high Fe average magnetic moment, ~ 3.2 B/Fe at room temperature will be performed by P3. Objective 2.2 Functional characterisation of hydrophilic MNPs Task 2.2.1 Magnetic properties analysis of -NH2 or -COOH MNPs, performed by P3. Task 2.2.2 Atomic Force Microscopy characterization of magnetite particles and MNPs with amino or carboxyl groups. This task will be performed by P4. Task 2.2.3 Magnetic characterization by magneto-optical Kerr effect: determination of hysteresis loops. This task will be performed by P3. Task 2.2.4 Micromagnetic characterization by Magnetic Force Microscopy, performed by P3. Task 2.2.5 Dimensional analysis and zeta potential study of magnetite particles and MNPs conjugates. This task will be performed by P3 + P4. Objective 2.3 Magnetic activation of NPs and passive intralesional delivery Task 2.3.1 Theoretical modeling and quantitation of thermal transfer to the environment will be performed initially. This task will be performed by P3 + P4. Task 2.3.2 Testing thermal dissipation will be assessed by means of an infrared pyrometer inside a special testing cell constructed with controlled atmosphere. Thermomagnetic measurements will also be carried by P3. Task 2.3.3 Testing as-prepared and functionalized NPs by X-ray photoelectron spectroscopy will be finally performed: including surface properties and NPs-ligand chemical interactions. This task will be performed by P4. Task 2.3.4 Passive difussion of the MNPs will be tested through injection into murine models tumor xenografts, normal pig liver and pancreas, as well as human tissue explants from HCC and PAC patients. Magnetic activation will be triggered through an external magnetic field, while the effects will be followed by the size of the ablated zone during follow-up. This task will be performed by all partners (CO, P1 to P4). Objective 2.4 Assessment of therapeutic effects (apoptosis / necrosis) Task 2.4.1 Pathology asessment of apoptosis will be accomplished through the immunohistochemical study of heat shock proteins (HSP-70 and HSP-90), whose expression following heat exposure might influence the extent of protein loss and degradation. Other markers for apoptosis which we will follow are interleukin 1 (IL-1) and tumor necrosis factor  (TNF). Task 2.4.2 Pathology asessment of necrosis will imply classical histological stains, as well as special immunostainings performed with polyclonal anti-ssDNA antibodies, anti-mithocondrial 113-1 antibody and mithocondrial enzyme cytochrome c oxidase subunit I antibody. Objective 2.5 MNP validation using the nano-ablation tester Task 2.3.1 Setup of the RFA tester will be performed by P2 + P3 Task 2.3.2 Calibration of the RFA tester on model surfaces and liquids with known emissivity Task 2.3.3 Definition of a validation protocol to be applied in case of MNPs

9 luni

Definition of experimental models

0 EUR
Obiective
1.1 Definition and protocol of the murine models (HCC + PAC): UMCV + UMFCN 1.2 Definition and protocol of the pig model: UMCV + UMFCN 1.3 Protocol for surgical tissue explants (HCC + PAC patients): UMCV + UMFCN 1.4 Report on the synthesis of magnetite-NH2 or COOH conjugates: ICMPP 1.5 Design of the nano-ablation tester: INCDFM
Activități
Objective 1.1 Definition and protocol of the murine models (HCC + PAC) Task 1.1.1 Definition of the HCC and PAC murine models for MNP hyperthermia will be based on xenografts using lab cultured HCC or PAC cell lines injections in severely immune deficient or nude mice, athymic (nu -/-) mice. Orthotopic implantations in the liver has advantages, including the possibility of testing in fibrotic livers (CCl4 model). This task will be accomplished by CO + P1, through common usage of small animal facilities in UMF Craiova and UMF Cluj-Napoca, which allow hosting of mice populations in baseline conditions of temperature, humidity, day-night cycle, etc. Task 1.1.2 Longitudinal monitoring of MNP treatment effects will be performed with contrastenhanced ultrasound (CEUS), based on 2nd generation microbubble contrast agents (SonoVue) in order to quantify angiogenesis changes during hyperthermia. Targeted US microbubbles probing VEGFR2 will be also used for monitoring of the tumors during MNP hyperthermia, through usage of specific contrast agents (Visistar VEGFR2). This task will be accomplished by CO + P1, because both centers (UMFCV + UMFCN) are equipped with high frequency state-of-the-art small animal ultrasound systems, with specific software used for quantification of angiogenesis. The procedures will be optimised based on the input from P3 + P4, related to the type and dosage of MNPs. PN-II-PT-PCCA-2011-3 24 Objective 1.2 Definition and protocol of the pig model Task 1.2.1 Experimental setting for transabdominal US-guided procedures (liver), as well as EUS-guided procedures (pancreas) in the pig model. Monitoring of the pigs for a period of 4 weeks will be accomplished through clinical & ultrasound examination, followed by necropsy. This task will be accomplished by CO + P1, through common usage of animal facilities in UMF Craiova and UMF Cluj-Napoca, which allow hosting in special boxes and monitoring of pig populations for at least 2 weeks. The procedures will be optimised based on the input from P2 related the RFA needle effects, as well as from P3 + P4 related to the type and dosage of MNPs. Objective 1.3 Protocol for surgical tissue explants (both HCC and PAC patients) Task 1.3.1 Identification of the clinical chain of harvesting HCC and PAC tissue explants from surgery, as well as the protocol of RFA and MNP hyperthermic treatment. Monitoring of necrosis will be accomplished by pathology examination (including apoptosis and necrosis evaluation). This task will be accomplished by CO + P1, optimised based on the input from P2 related the RFA needle effects, as well as from P3 + P4 related to the type and dosage of MNPs. Objective 1.4 Report on the synthesis of magnetite-NH2 or -COOH conjugates Task 1.4.1 Identification of the cheapest and easiest method for synthesis and preparation of controlled monodispersed MNPs. Hydrophilic magnetic particles are used for biomedical applications and therefore MNPs must be stable in saline solutions at physiological pH. This task will be accomplished by P4, based on previous experience and patents. Objective 1.5 Design of the nano-ablation tester Task 1.5.1 This tester will be based on IR monitoring of a cell containing MNP in fluides or of surfaces supporting MNPs, subject to RF activation. Both P2 + P3 will perform the task.