Development of green nanoparticles through solvent-free techniques and of alternative in vitro models for their validation

Introduction: Recently, nanotechnology has been exploited for the development of drug-delivery systems. Nanoparticle (NP) brought many advantages in cancer therapy, such as targeting, reduction of side, and improved biodistribution. Traditionally, NPs were synthesized using environmental unsustainable methods, which frequently employed organic solvents. The aim of this work is the preparation of solvent-free NPs for proteins and RNA delivery to melanoma, which will be tested on a 3D-printed model of metastatic melanoma model (under development). Methods: Two solvent-free techniques were investigated to synthetize green NPs: i) Antibody-loaded Chitosan (CS) NPs obtained by ionic gelation and ii) siRNA-loaded phosphate-poly(allylamine-hydrochloride) (PAH) NPs, obtained through electrostatic self-assembly. NPs were characterized in terms of size (DLS), loading efficiency, cell compatibility, and platelet activation. Different compositions of a collagen-hyaluronic acid (C/HA) bioink were tested to find the optimal ratio in terms of printability, shape fidelity, and cell viability. Human fibroblast and human melanoma cells were embedded in the C/HA bioink to obtain a cellularized architecture. Cell viability in the gel (cell titer-blue) was assessed after 1, 4, and 7 days. Results: NPs were successfully obtained with both methods, achieving a size of 200 nm for CS NPs and 90 nm for PAH NPS. SEM and FACS analysis showed that PAH NPs did not trigger platelet activation, at any of the tested concentrations, while CS NPs did not induce activation at concentrations below 200 µg/mL. Nearly 100% of the siRNA was successfully complexed into PAH NPs, while CS NPs encapsulated nearly 40% of the loaded Ab. FACS analysis and confocal microscopy showed that NPs were able to significantly enhance siRNA and protein accumulation in cells. Printability tests revealed that the optimal C/HA ratio is 50:50 and viability tests and confocal microscopy analysis confirmed the hydrogel biocompatibility with the tested cell line. Conclusions: CS and PAH NPs of small size and good encapsulation efficiency were successfully obtained without organic solvents. NPs exhibited good cell compatibility and extremely low platelet activation. 3D bioprinted C/HA matrix was realized allowing the cells to grow in a physiological-like environment. The model will be further characterized and improved by including blood vessels, followed by investigation of NPs ability to penetrate within the tumor and to target metastatic cells. Acknowledgements: Carlotta Mattioda acknowledges PON "Ricerca e Innovazione" 2014-2020 Azione IV.R "dottorati su tematiche green" for co-financing her Ph.D scholarship.