Functionalized Graphene Oxide Thin Films for Anti-tumor Drug Delivery to Melanoma Cells.

Livia E. Sima,Cristian V. A. Munteanu,Felix Sima,Emanuel Axente,Irina Negut,Valentina Grumezescu,Stefana Orobeti,Gabriela Chiritoiu

doi:10.3389/fchem.2020.00184

Abstract

Since Graphene discovery, their associated derivate nanomaterials, Graphene Oxide (GO) and reduced-GO were in the forefront of continuous developments in bio-nano-technology due to unique physical-chemical properties. Although GO nano-colloids (GON) were proposed as drug release matrix for targeting cancer cells, there is still a concern regarding its cytotoxicity issues. In this study, we report on the fabrication of functional GON bio-coatings by Matrix-Assisted Pulsed Laser Evaporation (MAPLE) to be used as drug carriers for targeting melanoma cells. We first performed a thorough in vitro cytotoxicity assay for comparison between GON and protein functionalized GON coatings. As functionalization protein, Bovine Serum Albumin (BSA) was non-covalently conjugated to GO surface. Safe concentration windows were identified in cytotoxicity tests by live/dead staining and MTS assays for five different human melanoma cell lines as well as for non-transformed melanocytes and human dermal fibroblasts. Hybrid GON-BSA nano-scaled thin coatings incorporating Dabrafenib (DAB) and Trichostatin A (TSA) inhibitors for cells bearing BRAFV600E pathway activating mutation were assembled on solid substrates by MAPLE technique. We further demonstrated the successful immobilization for each drug-containing GON-BSA assembling systems by evaluating cellular BRAF activity inhibition and histone deacetylases activity blocking, respectively. DAB activity was proven by the decreased ERK phosphorylation in primary melanoma cells (SKmel28 BRAFV600E cell line), while TSA effect was evidenced by acetylated histones accumulation in cell's nuclei (SKmel23 BRAF WT cell line). In addition, melanoma cells exposed to GON-BSA coatings with compositional gradient of inhibitors evidenced a dose-dependent effect on target activity. Such functional bio-platforms could present high potential for cell-biomaterial interface engineering to be applied in personalized cancer therapy studies.

Highlights

Carbon-based nanomaterials are extensively used in various fields of nanoscience and nanotechnology due to their exclusive physicochemical properties
We demonstrated that GONB-DAB and GONB-Trichostatin A (TSA) composite coatings are effective against melanoma cells during in vitro assays
Bovine Serum Albumin (BSA) and anti-cancer drug functionalized GO nano-colloids (GON) nanomaterials were successfully immobilized on solid substrate platforms by Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique and tested with melanoma cells

Summary

INTRODUCTION

Carbon-based nanomaterials are extensively used in various fields of nanoscience and nanotechnology due to their exclusive physicochemical properties. The authors evidenced a high potential of cell proliferation for both coatings (with certain predominance for oxidized Levan) by in vitro colorimetric assays Extracellular matrix proteins, such as fibronectin (FN) and vitronectin were assembled as thin layers on solid substrates by MAPLE, while preserving their biological functions (Sima et al, 2011a,b). Non-covalent surface functionalization of GO nano-colloids (GON) with Bovine Serum Albumin (BSA) protein was carried out following the protocol described elsewhere (Mu et al, 2012). Cell Viability Assessment by Live/Dead Staining The evaluation of cells viability and cytotoxicity of the serial dilutions of nano-colloids as well as GON and GONB thin films synthesized by MAPLE was performed using a LIVE/DEAD Viability/Cytotoxicity Assay Kit (Life Technologies) that allowed the simultaneous identification of live vs dead cells by fluorescence assessment. The gradient coatings were examined using the TissueFAXSiPlus system (TissueGnostics, Vienna, Austria)

RESULTS

DISCUSSION

CONCLUSIONS

DATA AVAILABILITY STATEMENT