Development of solvent-free nanoparticles for the treatment of metastatic melanoma and validation through in vitro models: an integrated approach

Melanoma is the most aggressive and life-threatening form of skin cancer, causing more than 70% of skin cancer deaths. The metastatic stage represents the last step of melanoma progression but constitutes the major challenge to the development of effective treatments. Current available therapies are not definitive for advanced stage melanoma and often present limitations such as side effects and drug resistance. Therefore, to improve the overall clinical outcome there is still a need to develop more specific therapies for metastatic melanoma. Nanomaterials are promising tools for drug delivery, as they can be designed to overcome biological barriers, to selectively target cancer cells, and to effectively delivery therapeutic agents to the tumour. Unfortunately, the process for the approval of novel therapies involves the use of ethically debated animal models that fail to reproduce human carcinogenesis, physiology, and progression. For these reasons, the aim of this work is to develop a set of nanoparticles (NPs) for the delivery of therapies against metastatic melanoma, based on the combination of silencing RNA (siRNA) and protein drugs, and to design a three-dimensional (3D) model of melanoma for the evaluation of these nanocarriers. Chitosan NPs for monoclonal antibody (mAb) delivery were generated through an optimized green ionic gelation method employing sodium tripolyphosphate (TPP) as a crosslinker, using a model fluorescently labelled mAb. To further stabilize NPs in blood circulation, a polyethylene glycol (PEG) coating was added on the surface of the NPs. Results showed that mAb was successfully encapsulated in NPs: loading efficiency resulted in a range between 30% and 56% depending on the initial amount of mAb provided. A sustained release of the mAb over 48 hours was also achieved. NPs designed to support delivery of siRNA were made with phosphate-poly(allylamine hydrochloride) (PAH) through a solvent free, single step self-assembly process, using a model fluorescent siRNA. The resulting NPs displayed an high siRNA encapsulation (99,6±0,05%). Both NP formulations were tested against melanoma cell lines (SK-MEL-28 and A375) and fibroblasts (HFF-1), with no signs of toxicity, proving the biocompatibility of the employed materials. The efficacy of NPs mediated delivery, compared with that of free mAb and siRNA, showed a higher cellular internalization for all the NPs tested composition. For the melanoma model, 3D bioprinting technology was used to embed HFF-1 cells in a biomimetic collagen/ hyaluronic acid (C/HA) hydrogel. Several hydrogel compositions were tested to assess the characteristics of printing resolution, while ensuring good biocompatibility. Indeed, the results of live/dead staining experiments showed that cells survived the extrusion process. Moreover, these hydrogel matrices supported cells proliferation up to 28 day. Lastly, spheroids from A375 and SK-MEL-28 cells of defined size and cell number were generated and placed in the hydrogel matrix with or without fibroblasts to study stromal cells influence on tumour progression. In future studies, this 3D printed construct will be integrated with a fully endothelialized vessel and with additional compartments mimicking the metastatic sites, to enhance the level of mimicry.