Final Report Summary - MBCTHERANOSTICSPION (Development and in vivo validation of a SPION based Theranostic nanosystem for the treatment of Metastatic Breast Cancer.) 1. FINAL PUBLISHABLE SUMMARY REPORT.- A comprehensive summary overview of results, conclusions and the socio-economic impacts of the project.Cancer is one of the leading causes of death worldwide and breast cancer is the most commonly diagnosed cancer in women. Surgery and radiotherapy remain the cornerstones of treatment for early-stage cancer, but their practicability and efficacy are limited when cancer cells have already metastasized, a pathological feature that accounts for 90 % of human cancer deaths. In those cases, therapeutic decisions depend on the type of cancer, metastatic burden, patient symptoms, and several predictive factors but, most frequently, they involve systemic administration of antineoplastic agents. Unfortunately, the lack of selectivity of this treatment produces off-target toxicity in healthy cells, provoking undesired side effects to the patients. Besides this, there are other important limitations of this therapy, such as metastasis and multidrug resistance (MDR). The use of nanocarriers as cancer-targeted drug delivery systems could improve the overall pharmacological properties of commonly used chemotherapeutic agents and reduce the most of the drawbacks cited above. The biocompatibility and magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) make them ideal tools for a broad range of biomedical applications. The development of anticancer SPION-based diagnostic and therapeutic nanosystems holds a promise of revolutionizing biomedicine, helping to solve important unmet clinical needs. Such potential will only be fulfilled if appropriate methods for SPION production and for their subsequent tailoring to specific applications are established, something that remains challenging. In this project, we have developed a simple and low cost method to fabricate structurally and colloidally ultrastable, water soluble SPION/Silica NPs. We have used thermal decomposition to produce SPIONs of the highest quality, which were then silica-coated to confer to the whole hydrophilicity and great structural stability. Subsequent PEGylation was carried out to give them excellent colloidal stability, whilst still leaving reactive anchoring points for further functionalization. The correct composition and physicochemical properties of our PEGylated SPION/Silica NPs and their precursors were confirmed using a broad range of analytical techniques. On the other hand, Doxorubicin was loaded onto our PEGylated SPION/Silica NPs via hydrazone bond and their pH-sensitive controlled release was verified. Also, Cisplatin was loaded onto our PEGylated SPION/Silica NPs via a chemisorption mechanism. Moreover, interestingly we have investigated the chemisorption capacity of these NPs to load other metals, such as PET and SPECT radioisotopes (i.e. 68-Ga, 67-Ga, 111-In, 89-Zr), and a patent to protect intellectually this invention is currently under preparation.We have also demonstrated the good biocompatibility of the resulting PEGylated SPION/Silica NPs, as well as their in vitro suitabibility as T2 MRI contrast agents. We have also analyzed the colloidal stability of these NPs in aqueous solution, in the absence of salts or proteins, as well as in their presence. Our studies revealed that PEGylated SPION/Silica NPs have excellent colloidal stability under all conditions analyzed, and an article reporting these results has been published in Nanoscale. Later, we incubated tissue cultured cells with PEGylated NPs (with and without Folate conjugation) in static conditions, to test the selective FR-targeting and celullar uptake. In these assays, we could not observe great differences in terms of increased attachment to cells between both types of NPs. We believe that this is likely to be due to the fact that non-targeted NPs, in static conditions, can eventually interact with non-targeted cells. For this reason, we have planed to perform similar experiments in slide chambers through which NPs will be circulated at a constant flow rate (i.e. dynamic conditions). These assays are currently being optimized using funds obtained by the MC awardee to continue this project. Furthermore, we have developed a xenograft and syngeneic tumor model in mice implanting 4T1.2 breast cancer cells into their fourth mammary fat pad. Then, we have tested the toxicity of Doxorubicin treatment in this model mice. After that, we have examined the NPs biodistribution in healthy mice and its efficiency for selective targeting in tumor bearing mice. Our NPs have excellent features in terms of structural and colloidal stabilty, biocompatibility and suitability as T2-MRI contrast. However, our results revealed that the biodistribution of our SPION/Silica NPs has to be improved before it can be used for targeted drug delivery. Therefore, in order to enhance their behavior for this application, we have modified the surface chemistry of our NPs to decrease their hydrodynamic diameter. As an advance of this fruitful study, we have developed a new way to fabricate PEGylated SPION/Silica NPs with much better properties, both in terms of circulation times and T2-MRI contrast, and a patent with this invention and a manuscript reporting these results are currently under preparation. In summary, our work have provided a novel and robust method for the production of PEGylated SPION/Silica NPs that can be used as a tunable platform for the development of smart diagnostic and therapeutic nanosystems. Therefore, we are currently in better conditions to carry out the project as conceved and also have funds to extend it an additional year. In addition, our efforts to device ways of improving the pharmacokinetics of NPs like this, and their functionalization with this purpose have also provided an opportunity to publish an specialized review in a high impact journal.
