  Citation: Pennisi, R.; Musarra-Pizzo, M.; Velletri, T.; Mazzaglia, A.; Neri, G.; Scala, A.; Piperno, A.; Sciortino, M.T. Cancer-Related Intracellular Signalling Pathways Activated by DOXorubicin/Cyclodextrin- Graphene-Based Nanomaterials. Biomolecules 2022, 12, 63. https:// doi.org/10.3390/biom12010063 Academic Editors: Miguel Ángel Esteso and Ana Cristina Faria Ribeiro Received: 25 November 2021 Accepted: 30 December 2021 Published: 1 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article Cancer-Related Intracellular Signalling Pathways Activated by DOXorubicin/Cyclodextrin-Graphene-Based Nanomaterials Rosamaria Pennisi 1, * , Maria Musarra-Pizzo 1 , Tania Velletri 2,† , Antonino Mazzaglia 3 , Giulia Neri 1 , Angela Scala 1 , Anna Piperno 1 and Maria Teresa Sciortino 1, * 1 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, 98166 Messina, Italy; mmusarrapizzo@unime.it (M.M.-P.); giulia.neri@unime.it (G.N.); ascala@unime.it (A.S.); apiperno@unime.it (A.P.) 2 IFOM-Cogentech Società Benefit srl; via Adamello 16, 20139 Milan, Italy; tania.velletri@cogentech.it 3 Istituto per lo Studio dei Materiali Nanostrutturati, Consiglio Nazionale delle Ricerche (ISMN-CNR), V.le F. Stagno d’Alcontres 31, 98166 Messina, Italy; antonino.mazzaglia@cnr.it * Correspondence: rpennisi@unime.it (R.P.); maria.sciortino@unime.it (M.T.S.) † Local Unit: Scientific and Technological Park of Sicily, 95121 Catania, Italy. Abstract: In the last decade, nanotechnological progress has generated new opportunities to improve the safety and efficacy of conventional anticancer therapies. Compared with other carriers, graphene nanoplatforms possess numerous tunable functionalities for the loading of multiple bioactive com- pounds, although their biocompatibility is still a debated concern. Recently, we have investigated the modulation of genes involved in cancer-associated canonical pathways induced by graphene engineered with cyclodextrins (GCD). Here, we investigated the GCD impact on cells safety, the HEp-2 responsiveness to Doxorubicin (DOX) and the cancer-related intracellular signalling pathways modulated by over time exposure to DOX loaded on GCD (GCD@DOX). Our studies evidenced that both DOX and GCD@DOX induced p53 and p21 signalling resulting in G 0 /G 1 cell cycle arrest. A genotoxic behaviour of DOX was reported via detection of CDK (T14/Y15) activation and reduc- tion of Wee-1 expression. Similarly, we found a cleavage of PARP by DOX within 72 h of exposure. Conversely, GCD@DOX induced a late cleavage of PARP, which could be indicative of less toxic effect due to controlled release of the drug from the GCD nanocarrier. Finally, the induction of the autophagy process supports the potential recycling of DOX with the consequent limitation of its toxic effects. Together, these findings demonstrate that GCD@DOX is a biocompatible drug delivery system able to evade chemoresistance and doxorubicin toxicity. Keywords: graphene-based platform; cancer therapy; intracellular signalling pathway; doxorubicin 1. Introduction Over the past three decades, the application of nanotechnology in medicine has had a significant impact on cancer disease treatment. Several nanotherapeutics have been clini- cally approved to support conventional anticancer therapies, while many others are under clinical investigation [1–3]. These nanoparticles are characterised by sizes ranging from a few to a few hundreds of nanometres (1–500 nm) and superior surface properties that are able to improve the delivery and release of therapeutic agents at the site of action [4]. However, to date, the most important challenge in nanomedicine remains reducing tox- icity profile and enhancing therapeutic efficacy. Carbon-based nanomaterials, including nanodiamonds, fullerenes, carbon nanotubes, graphene, and carbon nanofibers, have been proposed as useful biological platforms due to their versatility, physical properties, and unique intracellular trafficking properties [5–10]. Surface functionalisation with polymers such as polyethylenimine (PEI, branched/linear), cationic dendrimers, glycopolymers, and poly amidoamine increases their solubility and intracellular uptake and stabilises their half-life in the cellular environment [11–14]. The engineering of graphene surfaces Biomolecules 2022, 12, 63. https://doi.org/10.3390/biom12010063 https://www.mdpi.com/journal/biomolecules