Mesenchymal stem cell (MSC) therapy has garnered attention for over a decade due to its potential in regenerative medicine, especially in the scope of spinal cord injury (SCI) repair (Cofano, 2019). MSC therapeutic potential has been attributed to MSC secretome, to their ability to recruit progenitor cells, and to replace deficient/dead cells (des Rieux, 2021). However, the clinical efficacy of MSC therapy has been limited by poor cell survival post-transplantation. Low engraftment, immune rejection, and cell death could explain that they have limited effect in time (Zhao, 2020).

MSCs can come from different origins and niches (De Berdt, 2022). Dental stem cells such as stem cells from the human apical papilla (SCAP) have a high therapeutic potential for central nervous system (CNS) repair as they originate from the neural crest, promote oligodendrocyte progenitor cell differentiation, and present immunomodulatory properties (Ruparel, 2013, De Berdt, 2015, Bianco, 2016, De Berdt, 2018, Kandalam, 2020).

Several strategies (e.g. encapsulation in hydrogels, genetic modification) have been used to increase cell survival post-transplantation (des Rieux, 2021). MSC administration as spheroids is one strategy to potentiate MSC therapeutic potential. By enforcing MSC close interactions and mimicking their microenvironment, MSC spheroids have increased immunomodulatory properties and stemness, and overall better survival post-transplantation (Cesarz and Tamama, 2016, Ezquer, 2018, Redondo-Castro, 2018). Among the different methods to form spheroids (Ryu et al., 2019), the magnetic field-induced approach relies on the association of cells coated with magnetic nanoparticles that, when subjected to a magnetic field, drives the cell association and spheroid formation (Baillargeon, 2019). This method has the advantages of being quick, reproducible, and scalable.

However, necrosis and immune rejection remain two main challenges that limit MSC spheroid therapeutic potential. Our objective was then to address the limited cell survival post-transplantation by combining two therapeutic agents, NecroX-5 and rapamycin, targeting on one hand necrosis and the other immune rejection. Reactive oxygen species (ROS) scavenger NecroX-5 (Kim, 2010) can reduce tissue necrosis post-transplant (Del Vento, 2019) and can promote macrophage phenotype shift from M1 to M2 (Nam, 2019). Rapamycin is a widely known immunosuppressive drug that reduces graft rejection (Li et al., 2014, Wang, 2017).

In this study, we hypothesized that by combining NecroX-5 and rapamycin with SCAP to form spheroids, we would reduce necrosis and immune rejection toward allogenic SCAP. Our objective was to provide the proof of concept that it was possible to associate closely MSCs and nanomedicines using a magnetic drive. Our strategy was to encapsulate NecroX-5 and rapamycin in magnetic nanocarriers and then to closely associate them with magnetized SCAP, using a magnet, to form hybrid spheroids. We aimed to obtain a localized effect of NecroX-5 and rapamycin directly on SCAP spheroids, increasing their residence time in vivo while limiting systemic exposition.

As we aimed to identify which type of nanocarrier was the most appropriate for our application, NecroX-5 and rapamycin were either encapsulated in polymeric nanoparticles (NP) (Del Vento, 2019, Ganipineni, 2018) or lipid nanocapsules (LNC) (Mwema, 2022, Heurtault, 2002), the former allowing a sustained release but with a lower encapsulation efficiency while the latter provides a high encapsulation efficiency but no controlled release. Both allowed the delivery of these non-soluble molecules without organic solvents and the co-encapsulation of Super Paramagnetic Iron Oxide Nanoparticles (SPIONs). SPION-loaded nanoparticles are susceptible to the magnetic field used to form spheroids and we thus hypothesized that they could be associated with SCAP during their formation. The combination of drug-loaded magnetic nanomedicines and SCAP is later referred to as hybrid spheroids.

The objective of this study was to develop and optimize hybrid spheroids as a tool to increase SCAP viability in vivo. First, we encapsulated NecroX-5 and rapamycin in two different nanocarriers and then assessed their association with SCAP to form hybrid spheroids. Then, the impact of this association was studied on SCAP spheroid viability and gene expression of immunomodulatory molecules. Once this novel hybrid system was characterized, in vivo SCAP viability was assessed in a xenograft model in immunocompetent mice, either as a cell suspension, spheroids, or hybrid spheroids.