In this NANOTAM project, we have designed and developed 10 new empty polymeric Therapeutic Nanostructures (TNs) which can be decorated with different targeting ligands and at the same time loaded with immunomodulatory or chemotherapeutic drugs. Hyaluronic Acid Therapeutic Nanostructures (HA-TNs) decorated with Mannose or with the t-LyP-1 peptide have been generated and evaluated for the in vitro and in vivo targeting of Tumor Associated Macrophages (TAM). These HA-TNs have been successfully engineered to encapsulate 2 chemotherapeutic drugs (Docetaxel, DCX and modified Gemcitabine, GEM-C14) and 1 immunostimulatory drug (Imiquimod, IMQ).
In vitro methodologies and protocols have been adapted and implemented for the toxicological and immunomodulatory evaluation of polymeric-TNs. Non-toxic TNs have been identified and evaluated for their ability to re-educate protumoral M2-like macrophages into antitumoral M1-like macrophages. The toxicity and induction of M1-polarization (ability to re-educate TAM) by HA-TNs loaded with DCX, GEM-C14 or IMQ were observed at similar concentrations in vitro.
Flow cytometry and confocal techniques have been used to evaluate the ability of HA-TNs decorated with different targeting ligands (Mannose or t-LyP-1) to be uptaken by M0, M1 or M2 macrophages in vitro, demonstrating a better ability for the Mannose-HA-TNs than the t-LyP-1-HA-TNs to be internalized by M2 macrophages.
In vivo biocompatibility/toxicological analysis of TNs, based on anatomical or behavioural observations, and biochemical analysis for IMQ, demonstrated no signs of toxicity for the intravenous administration (i.v.) of any TN.
In vivo biodistribution experiments have been performed by i.v. injection of DiR-loaded TNs into MN/MCA fibrosarcoma bearing mice. The IVIS evaluation demonstrated a higher accumulation of Mannose-HA-TNs and t-LyP-1-HA-TNs in the tumor, but also undesirable accumulation in liver and spleen. Of note, higher accumulation for the Mannose-HA-TNs was also observed in the lung.
Evaluation of TNs loaded with DCX, GEM-C14 or IMQ, decorated or not with t-LyP-1 peptide, using immunocompetent murine in vivo models of fibrosarcoma demonstrated a significant antitumoral effect only for the i.v. administration of GEM-C14-loaded-TNs. On-going experiments with Mannose-decorated-HA-TNs are expected to improve the amount of drugs reaching TAM in tumors and to achieve a higher antitumoral efficacy. The decoration of TNs with t-LyP-1 did not show improved antitumoral efficacy in our in vivo murine model (correlated with the poor ability observed in vitro for this targeting ligand to improve the recognition and uptake of TNs by TAM). Additional experiments with Mannose-HA-DCX-TNs must be performed modifying doses and times of injections. The use of IMQ free or loaded into TNs, administered intravenously or intratumorally, did not show significant antitumoral effect in fibrosarcoma bearing mice, however a significant immunostimulatory response was observed in some mice. Thus, we do not consider these HA-IMQ-TNs effective as monotherapy, however their immunostimulatory effect could be exploited for combination therapies (i.e. with immunogenic cell death activators).
Overall, the observations and ideas arising from our experimental research investigation have been published in two review manuscripts (1 under revision). Additional work is required for the preparation of our results for their publication in an original research manuscript and/or to explore the possibilities for patenting, exploitation and/or commercialization.
