Different academic results from the different WPs of the proposal have been obtained. Overall, the results can be described as follows:
WP 1: Several zinc-doped iron oxide NPs have been synthesized using thermal decomposition methods. The NPs have been characterized by TEM, DLS and UV-vis. The size of the different nanoparticles varies between 5 and 50 nm. Their magnetic characterization has also been performed, allowing the possibility to select the NPs with the best magnetic performance for following biological applications.
In addition, several strategies have been sought to develop water soluble NPs. The first one was to encapsulate the NPs with a polymer shell of poly(maleic anhydride-alt-1-octadecene). Unfortunately, the resulting NPs tend to precipitate after activation with NHS/EDC (for subsequent bioconjugation). We changed the solubilization method and encapsulate the hydrophobic NPs in the inner core of phospholipid-base micelles. This method has proven really useful in giving exceptional water soluble NPs. Alternatively, the use of a positively-charged polymer, PEI, was also assessed. Unfortunately, this method was not able to produce optimal amounts of water-soluble NPs for in vivo studies and had to be discarded.
For the bifunctional stimuli-responsive likers, a similar ligand with a disulfide bridge (reduction responsive mechanism) was prepared. The release mechanism of this ligand has also been tested both in vitro and in vivo and, the ligand seems to work properly.
WP 2: The ability to prepare polymers on Jurkat T cells (used as a model of human T cells) was assessed, but the viability of these cells was seriously compromised. In different experimental conditions tested, viabilities were always lower than 50%, which was not optimal for later in vivo studies. Additionally, it was thought that there is no need to incorporate polymers with multiple clickable groups because one reactive group per anchor site would be enough for the NPs to incorporate on T cells. This strategy has been validated and a method optimized to click NPs and also small molecules of different origin on the plasma membrane of T cells (Jurkat, human primary and CAR) T cells. Also, cancer cells have also shown ability to react with tetrazine-containing small molecules.
I also acquired experience working with immune cells and isolate T cells from PBMCs. Also, I am now able to work with cells for in vitro and in vivo studies.
WP 3: Different in vitro studies using confocal microscopy and flow cytometry were performed. These studies demonstrated that the prepared NPs can efficiently interact with T cells from different origin: Jurkat cells, human primary T cells, and CAR T cells.
WP 4: Extensive in vivo studies were performed to study the possibility of improving T cell accumulation in tumors using magnetic NPs and an external magnetic field.
Generally, NSG mice bearing two PC3-PSMA+ tumors (one on each flank) were used. Then, different formulations were injected intravenously and all the groups had a NdFeB magnet placed only on the left tumor. After different time points, animals were followed accordingly to the imaging technique being used.
Moreover, different radiolabeling strategies had to be optimized and implemented for NP, cell and NP-cell labelings.
During these 3 years, I have supervised 3 students in the two different groups I have been in. I also gave one Department talks and participated in two conferences, the last one the World Molecular Imaging Conference 2021 (WMIC 21) with a poster. Moreover, I participated in different publications and a recent review. I was also included in the 30 early career professionals selected as “Ones to Watch” for 2021 by the SNMMI.
