SP1 public results:
The development of new multicomponent (organic/inorganic) nanosized delivery systems has been established. These versatile systems allow the inclusion of an anticancer drug (e.g. chemotherapy agent, siRNA) together with the grafting of a targeting agent (i.e. monoclonal antibody). Moreover, due to the polymeric structure, imaging agents like fluorescent dyes or magnetic nanoparticles could also be included. All the developments of SP1 allowed assembling a library of components enabling the formation of novel theranostic tools, putting us one step closer to innovative anticancer-clinical applications. In particular, composite nano-carriers (micelles, coacervates and polymersomes) made with polymer and iron oxide nanoparticles, able to load drugs and to be decorated with antibodies. All together, these nanosystems are multifunctional by design (allowing drug delivery, MRI imaging, targeting, hyperthermia) and are the basis for developments made in all other SPs.
Among all the nanoparticles pre-developed in the very first part of the project, a selection of the “best” systems, has been performed based on objective criteria, listed hereafter, have been agreed upon by all partners and evaluated thanks to the work performed in different SPs: toxicity (SP2), stability in biological media (SP6), drug loading (SP1), scale up (SP4), magnetic properties (SP3, SP5), novelty (see Table).
SP2 public results:
- Testing of the physicochemical properties (size, agglomeration and aggregation measured by Dynamic Light Scattering – DLS) of NPs (provided by SP1) in their stock solution and in cell culture media has been performed. Among those investigated, we have identified a group of NPs for further analysis (in vitro and in vivo and ex vivo testing).
- In vitro and ex vivo cell culture systems representative of different body compartments such as lung (A549 cells and Human Skin Organ Culture (HSOC) model), skin (HaCaT and Balb/3T3), liver (HepG2), intestine (CaCo-2), immune system (TK6 human lymphoblasts) and kidney (MDCK), have been used to identify the basal cytotoxicity of selected NPs. Assays such as Colony Forming Efficiency (CFE) assay, MTT and Alamar Blue have been performed to evaluate the potential toxicity and the dose-response relationship exerted in vitro by NPs. In addition, the pro-oxidant and pro-inflammatory activity of NPs has been investigated.
- The potential toxicity exerted by magnetic NPs, which are suitable for hyperthermic treatments, has been investigated. The aim of the study was identifying the presence of short-lived and long-lived toxic intermediates generated by magnetic NPs after exposure to electric fields.
- The cytotoxicity studies have helped identifying promising NPs whose in vitro uptake and intracellular fate is currently under investigation in selected cell lines (activities performed in SP6).
- To evaluate the possible interaction of NPs with tissues and organs, in vivo experiments have been performed administering selected NPs to an animal model used for anti-tumoral efficacy evaluation.
SP3 public results:
At M48, the following results are available
- Functionalization of PCL-b-PEG copolymers with folic acid moieties and functionalization on amine groups with proteins were successfully achieved.
- RMSB developed a new and reproducible bone tumor model established in living mice with 2 breast cancer cell lines.
- High troughput MRI allowing in vivo bone tumor volumetry and measurment of tumor growth with very small tumor size (microliters).
- Two strategies of functionalization of PTMC-b-PGA polymerosomes with Her2 antibodies, one direct and one indirect, have been developed and optimized.
- The efficiency of targeting of Her2-functionalized NPs for BT474 cells has been demonstrated in-vitro and in-vivo with MRI control.
- PEG-PBLG, PTMC-b-PGA, NBRh1 and NBRh13 NPs have been successfully TC99m-radiolabelled and imaged in-vivo.
- PET system with field of view 5x5cm has been successfully tested for mice imaging.
- Evidence of targeting of MDA-MB-231 tumor cells by radiolabelled nanosystems has been observed.
- Coherent results between two different systems on two different models have been obtained and have to been confirmed assessing the reproducibility of each results
- GDR Imagiv, Nouveaux defis en Imagerie du petit animal, Marseille 28/09/2010 - 01/10/2010
- Synthesis, radiolabeling and in vivo imaging of two novel hybrid nanoparticles, Psimadas D, Ravagli C, Loudos G, Varvarigou A.D and Baldi G, 15th European Symposium on Radiopharmacy and Radiopharmaceuticals, April 8 - 11, 2010, Edinburgh, Scotland.
- Liposomes Research Days – Vancouver (02-05/08/10)
SP4 public results:
At month 36, SP4 subproject has produced the following optimized NPs formulations:
Four different polymeric materials were successfully applied for the preparation of organic drug loaded nanoparticles and four selected drugs (paclitaxel, doxorubicin, aplidin and siRNA agent) were used.
Four types of hybrid magnetite/polymeric NPs were optimized and produced at lab-scale up batches. The MRI properties of all of them appear promising as well as the hyperthermia results.
Four types of hybrid magnetite/polymeric, drug loaded NPs were optimized. The MRI properties of all of them appear promising as well as the hyperthermia results, which are very good for two of them.
Four different targeting models have been investigated: 1) EGFR for colon, 2) Her2 for ovarian and breast, 3) bisphosphonate for bone, 4) folate for general cancer targeting. Depending on the targeting model the best strategy for the obtainment of functionalised targeted NPs was developed either by functionalising the starting polymer matrix or the preformed nanocarrier
Preliminary in vitro and in vivo evaluation of the therapeutic activity of the organic NPs was performed. Additionally, the evaluation of the targeting capability has started.
The scale-up of selected polymers, inorganic magnetic cores and related NPs was perfomed and the material characterized and validated also for their stability under physiological conditions and under storage.
SP5 public results:
At M36 subproject 5 has produced the following major results:
Four hybrid systems containing magnetite core (NBRh13 and targeted NBRh13-hEGFR, BlockM and targeted BlockM-FA) were characterised in terms of hyperthermic, relaxometric, magnetic and dielectric properties and they all displayed very promising features as potential theranostic agents.
Seven different samples of hybrid systems based on magnetite were delivered for relaxivity and magnetic characterization only. Only two of these samples among the most promising were characterised in terms of hyperthermic properties.
The radio-labelling protocol of organic and hybrid NPs has been finalised with encouraging process yields, and several samples resulted very promising as contrast agents for SPECT technique, as confirmed by biodistribution and imaging studies.
Biodistribution and imaging studies in SPECT mode in normal mice have been accomplished for several organic and hybrid NPs. All samples resulted very promising as contrast agents for SPECT imaging.
The in vitro evaluation of the hyperthermic efficiency of hybrid not-loaded and not-targeted NPs have been carried out. Experiments on hyperthermic efficiency of targeted hybrid NPs (NBRh13_hEGFR) in comparison to not-targeted ones and on loaded Block-PTX-FA in comparison with the not loaded counterpart are currently being carried out.
The in vivo experiments are about to start since the delivery of tumor-bearing mice to partners for imaging experiments is about to be done.