Marco Orecchioni

Graphene as Cancer Theranostic Tool: Progress and Future Challenges

Nowadays cancer remains one of the main causes of death in the world. Current diagnostic techniques need to be improved to provide earlier diagnosis and treatment. Traditional therapy approaches to cancer are limited by lack of specificity and systemic toxicity. In this scenario nanomaterials could be good allies to give more specific cancer treatment effectively reducing undesired side effects and giving at the same time accurate diagnosis and successful therapy. In this context, thanks to its unique physical and chemical properties, graphene, graphene oxide (GO) and reduced graphene (rGO) have recently attracted tremendous interest in biomedicine including cancer therapy. Herein we analyzed all studies presented in literature related to cancer fight using graphene and graphene-based conjugates. In this context, we aimed at the full picture of the state of the art providing new inputs for future strategies in the cancer theranostic by using of graphene. We found an impressive increasing interest in the material for cancer therapy and/or diagnosis. The majority of the works (73%) have been carried out on drug and gene delivery applications, following by photothermal therapy (32%), imaging (31%) and photodynamic therapy (10%). A 27% of the studies focused on theranostic applications. Part of the works here discussed contribute to the growth of the theranostic field covering the use of imaging (i.e. ultrasonography, positron electron tomography, and fluorescent imaging) combined to one or more therapeutic modalities. We found that the use of graphene in cancer theranostics is still in an early but rapidly growing stage of investigation. Any technology based on nanomaterials can significantly enhance their possibility to became the real revolution in medicine if combines diagnosis and therapy at the same time. We performed a comprehensive summary of the latest progress of graphene cancer fight and highlighted the future challenges and the innovative possible theranostic applications.

Impact of carbon nanotubes and graphene on immune cells

It has been recently proposed that nanomaterials, alone or in concert with their specific biomolecular conjugates, can be used to directly modulate the immune system, therefore offering a new tool for the enhancement of immune-based therapies against infectious disease and cancer. Here, we revised the publications on the impact of functionalized carbon nanotubes (f-CNTs), graphene and carbon nanohorns on immune cells. Whereas f-CNTs are the nanomaterial most widely investigated, we noticed a progressive increase of studies focusing on graphene in the last couple of years. The majority of the works (56%) have been carried out on macrophages, following by lymphocytes (30% of the studies). In the case of lymphocytes, T cells were the most investigated (22%) followed by monocytes and dendritic cells (7%), mixed cell populations (peripheral blood mononuclear cells, 6%), and B and natural killer (NK) cells (1%). Most of the studies focused on toxicity and biocompatibility, while mechanistic insights on the effect of carbon nanotubes on immune cells are generally lacking. Only very recently high-throughput gene-expression analyses have shed new lights on unrecognized effects of carbon nanomaterials on the immune system. These investigations have demonstrated that some f-CNTs can directly elicitate specific inflammatory pathways. The interaction of graphene with the immune system is still at a very early stage of investigation. This comprehensive state of the art on biocompatible f-CNTs and graphene on immune cells provides a useful compass to guide future researches on immunological applications of carbon nanomaterials in medicine.

Molecular and Genomic Impact of Large and Small Lateral Dimension Graphene Oxide Sheets on Human Immune Cells from Healthy Donors.

Graphene oxide (GO) is attracting great interest in biomedical sciences. The impact of GO on immune cells is one fundamental area of study that is often overlooked, but critical in terms of clinical translation. This work investigates the effects of two types of thoroughly characterized GO sheets, different in their lateral dimension, on human peripheral immune cells provided from healthy donors using a wide range of assays. After evaluation of cell viability, the gene expression was analyzed, following GO exposure on 84 genes related to innate and adaptive immune responses. Exposure to GO small sheets was found to have a more significant impact on immune cells compared to GO large sheets, reflected in the upregulation of critical genes implicated in immune responses and the release of cytokines IL1β and TNFα. These findings were further confirmed by whole-genome microarray analysis of the impact of small GO sheets on T cells and monocytes. Activation in both cell types was underlined by the overexpression of genes such as CXCL10 and receptor CXCR3. Significant energy-dependent pathway modulation was identified. These findings can potentially pave the foundations for further design of graphene that can be used for immune modulation applications, for example in cancer immunotherapy.

Graphene and the immune system: Challenges and potentiality.

In the growing area of nanomedicine, graphene-based materials (GBMs) are some of the most recent explored nanomaterials. For the majority of GBM applications in nanomedicine, the immune system plays a fundamental role. It is necessary to well understand the complexity of the interactions between GBMs, the immune cells, and the immune components and how they could be of advantage for novel effective diagnostic and therapeutic approaches. In this review, we aimed at painting the current picture of GBMs in the background of the immune system. The picture we have drawn looks like a cubist image, a sort of Picasso-like portrait looking at the topic from all perspectives: the challenges (due to the potential toxicity) and the potentiality like the conjugation of GBMs to biomolecules to develop advanced nanomedicine tools. In this context, we have described and discussed i) the impact of graphene on immune cells, ii) graphene as immunobiosensor, and iii) antibodies conjugated to graphene for tumor targeting. Thanks to the huge advances on graphene research, it seems realistic to hypothesize in the near future that some graphene immunoconjugates, endowed of defined immune properties, can go through preclinical test and be successfully used in nanomedicine.

Immune cell impact of three differently coated lipid nanocapsules: pluronic, chitosan and polyethylene glycol

Lipid nanocapsules (NCs) represent promising tools in clinical practice for diagnosis and therapy applications. However, the NC appropriate functionalization is essential to guarantee high biocompatibility and molecule loading ability. In any medical application, the immune system-impact of differently functionalized NCs still remains to be fully understood. A comprehensive study on the action exerted on human peripheral blood mononuclear cells (PBMCs) and major immune subpopulations by three different NC coatings: pluronic, chitosan and polyethylene glycol-polylactic acid (PEG) is reported. After a deep particle characterization, the uptake was assessed by flow-cytometry and confocal microscopy, focusing then on apoptosis, necrosis and proliferation impact in T cells and monocytes. Cell functionality by cell diameter variations, different activation marker analysis and cytokine assays were performed. We demonstrated that the NCs impact on the immune cell response is strongly correlated to their coating. Pluronic-NCs were able to induce immunomodulation of innate immunity inducing monocyte activations. Immunomodulation was observed in monocytes and T lymphocytes treated with Chitosan-NCs. Conversely, PEG-NCs were completely inert. These findings are of particular value towards a pre-selection of specific NC coatings depending on biomedical purposes for pre-clinical investigations; i.e. the immune-specific action of particular NC coating can be excellent for immunotherapy applications.

Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH2) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.Understanding the interaction of nanomaterials and immune cells at the biomolecular level is of great significance in therapeutic applications. Here, the authors investigated the interaction of graphene oxide nanomaterials and several immune cell subpopulations using single-cell mass cytometry and genome-wide transcriptome analysis.

Few-Layer Graphene Kills Selectively Tumor Cells from Myelomonocytic Leukemia Patients

In the cure of cancer, a major cause of todays mortality, chemotherapy is the most common treatment, though serious frequent challenges are encountered by current anticancer drugs. We discovered that few-layer graphene (FLG) dispersions have a specific killer action on monocytes, showing neither toxic nor activation effects on other immune cells. We confirmed the therapeutic application of graphene on an aggressive type of cancer that is myelomonocytic leukemia, where the monocytes are in their malignant form. We demonstrated that graphene has the unique ability to target and boost specifically the necrosis of monocytic cancer cells. Moreover, the comparison between FLG and a common chemotherapeutic drug, etoposide, confirmed the higher specificity and toxicity of FLG. Since current chemotherapy treatments of leukemia still cause serious problems, these findings open the way to new and safer therapeutic approaches.

Immune compatible cystine-functionalized superparamagnetic iron oxide nanoparticles as vascular contrast agents in ultrasonography

Superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively investigated for many biomedical applications. A good quality functionalization that combines imaging goals with a high-level of biocompatibility remains one of the challenges for particle translation into medical practice. Here, we focus on a new functionalization of SPIONs with cystine (Cy-SPIONs). Cystine is able to make SPIONs stable and dispersible in water and in culture cell media. New insights are provided into the biological and immune effects of Cy-SPIONs with a wide variety of standard and molecular assays to evaluate cytotoxicity, cell activation, cytokine release and the expression of 84 genes related to immune responses. A good immune biocompatibility of Cy-SPIONs on primary immune cells was found. The great potential of Cy-SPIONs for further in vivo studies and as contrast agents for magnetic resonance imaging (MRI) is highlighted. In addition, we also exploited ultrasonography, since it is a safer, less expensive and common imaging technology. The good echogenic properties of Cy-SPIONs in water and in whole blood are shown, both in vitro and in a phantom vein for bloodstream simulations. Our results open up a new scenario for future applications of cystine-functionalized SPIONs as immune-compatible ultrasound and MRI contrast agents.

Carbon nanomaterials as contrast agents for breast cancer diagnosis and therapy

Nanotechnology is the promise to fight breast cancer (BC) more specifically and effectively [1]. In this context carbon nanomaterials (CNs) have attracted the scientific community and the public interest [2]. Common modalities for BC diagnosis are ultrasonography (US) and magnetic resonance imaging (MRI). US is the most useful modality in the evaluation of palpable BC masses that are mammographically occult in women younger than 30’s. Here we show CNs as highly and long lasting echogenic materials. Experiments on swine models confirmed that CNs are clearly visible under US and didn’t exert toxicity. In the current market dual-imaging agents are missed; here we also demonstrate the immune-compatibility and high echogenic properties in water and in whole blood of cysteine functionalized super paramagnetic nanoparticles (CY-SPION), well-known MRI agents [3]. Thanks to these findings, and the ability to load CNs to many moieties [4-6], we propose dual-contrast agents, CNs-CY-SPION conjugates, to improve BC diagnosis. Future perspectives is to conjugate CN-SPION to targeted drugs against BC. In summary, we lay the foundations for novel contrast agents, for therapy and multimodal diagnosis of BC, combining high imaging performances with unique potential therapeutic applications, such as specific targeting capabilities, drug delivery, immunotherapy and hyperthermia.

Molecular impact of graphene oxide with different shape dimension on human immune cells

In the last few years, there has been enormous interest in graphene oxide (GO) for its wide variety of applications[1]. However, for any medical application, the immune system-impact of GO still remain to be fully understood. Moreover, the modulation of immune cells mediated by nanomaterials could be interesting also in immunotheraphy applications[2]. Indeed, nanomaterials and more in general nanotechnology can enhance the efficacy of immunostimulatory small molecules and biologics by altering their co-localization, biodistribution, and release kinetics[3]. Following these aims we focused on the molecular effects of two GOs, different for lateral size dimensions, on human peripheral blood mononuclear cells (PBMCs). GOs were fully characterized then, we performed a wide range of standard assays looking at cell viability, cell activation and multiple cytokines secretion. We characterized the molecular impact of GOs on 84 genes immune-response-related. Additionally, a whole genome analysis was conducted on T cells and monocytes as representative of the innate and adaptive immune responses. In Figure ​Figure11 TEM and AFM characterization of GO-Small (140 nm) and GO-Large (4mm). We did not detect any toxicity in GO PBMCs treated samples. The 84 gene expression analysis evidenced a clear dimension-dependent impact of GOs on cell activation (Figure ​(Figure2).2). In particular, the GO-Small modulated 16 genes (Fold Regulation >4) compared to only 5 of GO-Large (in red in Figure ​Figure22 C). Action confirmed also by cytokine analysis (Figure ​(Figure22 D). These evidences were also confirmed by microarray analysis on T and monocytes cell lines. GO-Small impact the immune cell activation, underlined by the over expression of many pathways such as leukocyte chemotaxis pathway (Figure ​(Figure3),3), genes such as CXCL10 ligand pathway and CXCR3 receptor (Figure ​(Figure3,3, red box). Moreover, we found a strong action on cell metabolism with a down-regulation on energetic pathways such as oxidative-phosphorylation pathway in both cell types (data not shown). Our work represents a comprehensive molecular-characterization of different sized GOs on immune cells giving crucial information for the chemical and physical design of graphene for biomedical applications i.e. as a new possible drug delivery systems and nanoimmunotherapy tools. Figure 1 Figure 2 Figure 3