Photocrosslinked Co-Assembled Amino Acid Nanoparticles for Controlled Chemo/Photothermal Combined Anticancer Therapy.

Nanostructures formed from the self-assembly of amino acids are promising materials in many fields, especially for biomedical applications. However, their low stability resulting from the weak noncovalent interactions between the amino acid building blocks limits their use. In this work, nanoparticles co-assembled by fluorenylmethoxycarbonyl (Fmoc)-protected tyrosine (Fmoc-Tyr-OH) and tryptophan (Fmoc-Trp-OH) are crosslinked by ultraviolet (UV) light irradiation. Two methods are investigated to induce the dimerization of tyrosine, irradiating at 254 nm or at 365 nm in the presence of riboflavin as a photo-initiator. For the crosslinking performed at 254 nm, both Fmoc-Tyr-OH and Fmoc-Trp-OH generate dimers. In contrast, only Fmoc-Tyr-OH participates in the riboflavin-mediated dimerization under irradiation at 365 nm. The participation of both amino acids in forming the dimers leads to more stable crosslinked nanoparticles, allowing also to perform further chemical modifications for cancer applications. The anticancer drug doxorubicin (Dox) is adsorbed onto the crosslinked nanoparticles, subsequently coated by a tannic acid-iron complex, endowing the nanoparticles with glutathione-responsiveness and photothermal properties, allowing to control the release of Dox. A remarkable anticancer efficiency is obtained in vitro and in vivo in tumor-bearing mice thanks to the combined chemo- and photothermal treatment.

Unveiling Liquid-Phase Exfoliation of Graphite and Boron Nitride Using Fluorescent Dyes Through Combined Experiments and Simulations.

Liquid-phase exfoliation (LPE) in aqueous solutions provides a simple, scalable, and green approach to produce 2D materials. By combining atomistic simulations with exfoliation experiments, the interaction between a surfactant and a 2D layer at the molecular scale can be better understood. In this work, two different dyes, corresponding to rhodamine B base (Rbb) and to a phenylboronic acid BODIPY (PBA-BODIPY) derivative, are employed as dispersants to exfoliate graphene and hexagonal boron nitride (hBN) through sonication-assisted LPE. The exfoliated 2D sheets, mostly as few-layers, exhibit good quality and high loading of dyes. Using molecular dynamics (MD) simulations, the binding free energies are calculated and the arrangement of both dyes on the layers are predicted. It has been found that the dyes show a higher affinity toward hBN than graphene, which is consistent with the higher yields of exfoliated hBN. Furthermore, it is demonstrated that the adsorption behavior of Rbb molecules on graphene and hBN is quite different compared to PBA-BODIPY.

Celebrating Maurizio Prato's Passion, Talent and Imagination.

This Editorial introduces a Special Collection of papers dedicated to Maurizio Prato, featuring prominent examples of his team's efforts to integrate complex frontier research with pioneering achievements in the field of carbon nanostructures and molecular nanoscience.

Synthesis of Visible Light Excitable Carbon Dot Phosphor-Al2 O3 Hybrids for Anti-Counterfeiting and Information Encryption.

The preparation of room temperature phosphorescent carbon dots still faces great challenges, especially in the case of carbon dots endowed of visible-light excitable room temperature phosphorescence (RTP). To date, a limited number of substrates have been exploited to synthesize room temperature phosphorescent carbon dots, and most of them can emit RTP only in solid state. Here, the synthesis of a composite obtained from the calcination of green carbon dots (g-CDs) blended with aluminum hydroxide (Al(OH)3 ) is reported. The resultant hybrid material g-CDs@Al2 O3 exhibits blue fluorescence and green RTP emissions in an on/off switch process at 365 nm. Notably, this composite manifests strong resistance to extreme acid and basic conditions up to 30 days of treatment. The dense structure of Al2 O3 formed by calcination contributes to the phosphorescent emission of g-CDs. Surprisingly, g-CDs@Al2 O3 can also emit yellow RTP under irradiation with white light. The multicolor emissions can be employed for anti-counterfeiting and information encryption. This work provides a straightforward approach to produce room temperature phosphorescent carbon dots for a wide range of applications.

Mechanisms of Radical Formation on Chemically Modified Graphene Oxide under Near Infrared Irradiation.

In this work, the mechanisms of radical generation on different functionalized graphene oxide (GO) conjugates under near-infrared (NIR) light irradiation are investigated. The GO conjugates are designed to understand how chemical functionalization can influence the generation of radicals. Both pristine and functionalized GO are irradiated by a NIR laser, and the production of different reactive oxygen species (ROS) is investigated using fluorimetry and electron paramagnetic resonance to describe the type of radicals present on the surface of GO. The mechanism of ROS formation involves a charge transfer from the material to the oxygen present in the media, via the production of superoxide and singlet oxygen. Cytotoxicity and effects of ROS generation are then evaluated using breast cancer cells, evidencing a concentration dependent cell death associated to the heat and ROS. The study provides new hints to understand the photogeneration of radicals on the surface of GO upon near infrared irradiation, as well as, to assess the impact on these radicals in the context of a combined drug delivery system and phototherapeutic approach. These discoveries open the way for a better control of phototherapy-based treatments employing graphene-based materials.

Functional Graphene for Peritumoral Brain Microenvironment Modulation Therapy in Glioblastoma.

Peritumoral brain invasion is the main target to cure glioblastoma. Chemoradiotherapy and targeted therapies fail to combat peritumoral relapse. Brain inaccessibility and tumor heterogeneity explain this failure, combined with overlooking the peritumor microenvironment. Reduce graphene oxide (rGO) provides a unique opportunity to modulate the local brain microenvironment. Multimodal graphene impacts are reported on glioblastoma cells in vitro but fail when translated in vivo because of low diffusion. This issue is solved by developing a new rGO formulation involving ultramixing during the functionalization with polyethyleneimine (PEI) leading to the formation of highly water-stable rGO-PEI. Wide mice brain diffusion and biocompatibility are demonstrated. Using an invasive GL261 model, an anti-invasive effect is observed. A major unexpected modification of the peritumoral area is also observed with the neutralization of gliosis. In vitro, mechanistic investigations are performed using primary astrocytes and cytokine array. The result suggests that direct contact of rGO-PEIUT neutralizes astrogliosis, decreasing several proinflammatory cytokines that would explain a bystander tumor anti-invasive effect. rGO also significantly downregulates several proinvasive/protumoral cytokines at the tumor cell level. The results open the way to a new microenvironment anti-invasive nanotherapy using a new graphene nanomaterial that is optimized for in vivo brain delivery.

Lung Persistence, Biodegradation, and Elimination of Graphene-Based Materials are Predominantly Size-Dependent and Mediated by Alveolar Phagocytes.

Graphene-based materials (GBMs) have promising applications in various sectors, including pulmonary nanomedicine. Nevertheless, the influence of GBM physicochemical characteristics on their fate and impact in lung has not been thoroughly addressed. To fill this gap, the biological response, distribution, and bio-persistence of four different GBMs in mouse lungs up to 28 days after single oropharyngeal aspiration are investigated. None of the GBMs, varying in size (large versus small) and carbon to oxygen ratio as well as thickness (few-layers graphene (FLG) versus thin graphene oxide (GO)), induce a strong pulmonary immune response. However, recruited neutrophils internalize nanosheets better and degrade GBMs faster than macrophages, revealing their crucial role in the elimination of small GBMs. In contrast, large GO sheets induce more damages due to a hindered degradation and long-term persistence in macrophages. Overall, small dimensions appear to be a leading feature in the design of safe GBM pulmonary nanovectors due to an enhanced degradation in phagocytes and a faster clearance from the lungs for small GBMs. Thickness also plays an important role, since decreased material loading in alveolar phagocytes and faster elimination are found for FLGs compared to thinner GOs. These results are important for designing safer-by-design GBMs for biomedical application.

Graphene Oxide-BODIPY Conjugates as Highly Fluorescent Materials.

Covalent functionalization of graphene oxide (GO) with boron dipyrromethenes (BODIPYs) was achieved through a facile synthesis, affording two different GO-BODIPY conjugates where the main difference lies in the nature of the spacer and the type of bonds between the two components. The use of a long but flexible spacer afforded strong electronic GO-BODIPY interactions in the ground state. This drastically altered the light absorption of the BODIPY structure and impeded its selective excitation. In contrast, the utilisation of a short, but rigid spacer based on boronic esters resulted in a perpendicular geometry of the phenyl boronic acid BODIPY (PBA-BODIPY) with respect to the GO plane, which enables only minor electronic GO-BODIPY interactions in the ground state. In this case, selective excitation of PBA-BODIPY was easily achieved, allowing to investigate the excited state interactions. A quantitative ultrafast energy transfer from PBA-BODIPY to GO was observed. Furthermore, due to the reversible dynamic nature of the covalent GO-PBA-BODIPY linkage, some PBA-BODIPY is free in solution and, hence, not quenched from GO. This resulted in a weak, but detectable fluorescence from the PBA-BODIPY that will allow to exploit GO-PBA-BODIPY for slow release and imaging purposes.

Self-assembly of amphiphilic amino acid derivatives for biomedical applications.

Amino acids are one of the simplest biomolecules and they play an essential role in many biological processes. They have been extensively used as building blocks for the synthesis of functional nanomaterials, thanks to their self-assembly capacity. In particular, amphiphilic amino acid derivatives can be designed to enrich the diversity of amino acid-based building blocks, endowing them with specific properties and/or promoting self-assembly through hydrophobic interactions, hydrogen bonding, and/or π-stacking. In this review, we focus on the design of various amphiphilic amino acid derivatives able to self-assemble into different types of nanostructures that were exploited for biomedical applications, thanks to their excellent biocompatibility and biodegradability.

2D Materials and Primary Human Dendritic Cells: A Comparative Cytotoxicity Study.

Human health can be affected by materials indirectly through exposure to the environment or directly through close contact and uptake. With the ever-growing use of 2D materials in many applications such as electronics, medical therapeutics, molecular sensing, and energy storage, it has become more pertinent to investigate their impact on the immune system. Dendritic cells (DCs) are highly important, considering their role as the main link between the innate and the adaptive immune system. By using primary human DCs, it is shown that hexagonal boron nitride (hBN), graphene oxide (GO) and molybdenum disulphide have minimal effects on viability. In particular, it is evidenced that hBN and GO increase DC maturation, while GO leads to the release of reactive oxygen species and pro-inflammatory cytokines. hBN and MoS2 increase T cell proliferation with and without the presence of DCs. hBN in particular does not show any sign of downstream T cell polarization. The study allows ranking of the three materials in terms of inherent toxicity, providing the following trend: GO > hBN ≈ MoS2 , with GO the most cytotoxic.

Recent Advances in 2D Material-Mediated Immuno-Combined Cancer Therapy.

In the last years, cancer immunotherapy has started to attract a lot of attention, becoming one of the alternatives in the clinical treatment of cancer. Indeed, one of the advantages of immunotherapy is that both primary and distant tumors can be efficiently eradicated through a triggered immune response. Due to their large specific surface area and unique physicochemical properties, 2D materials have become popular in cancer immunotherapy, especially as efficient drug carriers. They have been also exploited as photothermal platforms, chemodynamic agents, and photosensitizers to further enhance the efficacy of the therapy. In this review, the focus is on the recent development of 2D materials as new tools to combine immunotherapy with chemotherapy, photothermal therapy, photodynamic therapy, chemodynamic therapy, radiotherapy, and radiodynamic therapy. These innovative synergistic approaches intend to go beyond the classical strategies based on a simple delivery function of immune modulators by nanomaterials. Furthermore, the effects of the 2D materials themselves and their surface properties (e.g., chemical modification and protein corona formation) on the induction of an immune response will be also discussed.

Nanobiosensor Reports on CDK1 Kinase Activity in Tumor Xenografts in Mice.

Probing the dynamics and quantifying the activities of intracellular protein kinases that coordinate cell growth and division and constitute biomarkers and pharmacological targets in hyperproliferative and pathological disorders remain a challenging task. Here engineering and characterization of a nanobiosensor of the mitotic kinase CDK1, through multifunctionalization of carbon nanotubes with a CDK1-specific fluorescent peptide reporter, are described. This original reporter of CDK1 activity combines the sensitivity of a fluorescent biosensor with the unique physico-chemical and biological properties of nanotubes for multifunctionalization and efficient intracellular penetration. The functional versatility of this nanobiosensor enables implementation to quantify CDK1 activity in a sensitive and dose-dependent fashion in complex biological environments in vitro, to monitor endogenous kinase in living cells and directly within tumor xenografts in mice by fluorescence imaging, thanks to a ratiometric quantification strategy accounting for response relative to concentration in space and in time.

Biodegradation of graphene materials catalyzed by human eosinophil peroxidase.

Understanding the biodegradability of graphene materials by the action of oxidative enzymes secreted by immune cells is essential for developing applicable biomedical products based on these materials. Herein, we demonstrate the biodegradation of graphene oxide (GO) by recombinant eosinophil peroxidase (EPO) enzyme extracted from human eosinophils in the presence of a low concentration of hydrogen peroxide and NaBr. We compared the degradation capability of the enzyme on three different GO samples containing different degrees of oxygen functional groups on their graphenic lattices. EPO succeeded in degrading the three tested GO samples within 90 h treatment. Raman spectroscopy and transmission electron microscopy analyses provided clear-cut evidence for the biodegradation of GO by EPO catalysis. Our results provide more insight into a better understanding of the biodegradation of graphene materials, helping the design of future biomedical products based on these carbon nanomaterials.

Degradation-by-design: how chemical functionalization enhances the biodegradability and safety of 2D materials.

A large number of graphene and other 2D materials are currently used for the development of new technologies, increasingly entering different industrial sectors. Interrogating the impact of such 2D materials on health and environment is crucial for both modulating their potential toxicity in living organisms and eliminating them from the environment. In this context, understanding if 2D materials are bio-persistent is mandatory. In this review we describe the importance of biodegradability and decomposition of 2D materials. We initially cover the biodegradation of graphene family materials, followed by other emerging classes of 2D materials including transition metal dichalcogenides and oxides, Xenes, Mxenes and other non-metallic 2D materials. We explain the role of defects and functional groups, introduced onto the surface of the materials during their preparation, and the consequences of chemical functionalization on biodegradability. In strong relation to the chemistry on 2D materials, we describe the concept of "degradation-by-design" that we contributed to develop, and which concerns the covalent modification with appropriate molecules to enhance the biodegradability of 2D materials. Finally, we cover the importance of designing new biodegradable 2D conjugates and devices for biomedical applications as drug delivery carriers, in bioelectronics, and tissue engineering. We would like to highlight that the biodegradation of 2D materials mainly depends on the type of material, the chemical functionalization, the aqueous dispersibility and the redox potentials of the different oxidative environments. Biodegradation is one of the necessary conditions for the safe application of 2D materials. Therefore, we hope that this review will help to better understand their biodegradation processes, and will stimulate the chemists to explore new chemical strategies to design safer products, composites and devices containing 2D materials.

How macrophages respond to two-dimensional materials: a critical overview focusing on toxicity.

With wider use of graphene-based materials and other two-dimensional (2 D) materials in various fields, including electronics, composites, biomedicine, etc., 2 D materials can trigger undesired effects at cellular, tissue and organ level. Macrophages can be found in many organs. They are one of the most important cells in the immune system and they are relevant in the study of nanomaterials as they phagocytose them. Nanomaterials have multi-faceted effects on phagocytic immune cells like macrophages, showing signs of inflammation in the form of pro-inflammatory cytokine or reactive oxidation species production, or upregulation of activation markers due to the presence of these foreign bodies. This review is catered to researchers interested in the potential impact and toxicity of 2 D materials, particularly in macrophages, focusing on few-layer graphene, graphene oxide, graphene quantum dots, as well as other promising 2 D materials containing molybdenum, manganese, boron, phosphorus and tungsten. We describe applications relevant to the growing area of 2 D materials research, and the possible risks of ions and molecules used in the production of these promising 2 D materials, or those produced by the degradation and dissolution of 2 D materials.

Comparative Effects of Graphene and Molybdenum Disulfide on Human Macrophage Toxicity.

Graphene and other 2D materials, such as molybdenum disulfide, have been increasingly used in electronics, composites, and biomedicine. In particular, MoS2 and graphene hybrids have attracted a great interest for applications in the biomedical research, therefore stimulating a pertinent investigation on their safety in immune cells like macrophages, which commonly engulf these materials. In this study, M1 and M2 macrophage viability and activation are mainly found to be unaffected by few-layer graphene (FLG) and MoS2 at doses up to 50 µg mL-1 . The uptake of both materials is confirmed by transmission electron microscopy, inductively coupled plasma mass spectrometry, and inductively coupled plasma atomic emission spectroscopy. Notably, both 2D materials increase the secretion of inflammatory cytokines in M1 macrophages. At the highest dose, FLG decreases CD206 expression while MoS2 decreases CD80 expression. CathB and CathL gene expressions are dose-dependently increased by both materials. Despite a minimal impact on the autophagic pathway, FLG is found to increase the expression of Atg5 and autophagic flux, as observed by Western blotting of LC3-II, in M1 macrophages. Overall, FLG and MoS2 are of little toxicity in human macrophages even though they are found to trigger cell stress and inflammatory responses.

Intracerebral Injection of Graphene Oxide Nanosheets Mitigates Microglial Activation Without Inducing Acute Neurotoxicity: A Pilot Comparison to Other Nanomaterials.

Carbon-based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial-based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain.

Toward High-Dimensional Single-Cell Analysis of Graphene Oxide Biological Impact: Tracking on Immune Cells by Single-Cell Mass Cytometry.

Considering the potential exposure to graphene, the most investigated nanomaterial, the assessment of the impact on human health has become an urgent need. The deep understanding of nanomaterial safety is today possible by high-throughput single-cell technologies. Single-cell mass cytometry (cytometry by time-of flight, CyTOF) shows an unparalleled ability to phenotypically and functionally profile complex cellular systems, in particular related to the immune system, as recently also proved for graphene impact. The next challenge is to track the graphene distribution at the single-cell level. Therefore, graphene oxide (GO) is functionalized with AgInS2 nanocrystals (GO-In), allowing to trace GO immune-cell interactions via the indium (115 In) channel. Indium is specifically chosen to avoid overlaps with the commercial panels (>30 immune markers). As a proof of concept, the GO-In CyTOF tracking is performed at the single-cell level on blood immune subpopulations, showing the GO interaction with monocytes and B cells, therefore guiding future immune studies. The proposed approach can be applied not only to the immune safety assessment of the multitude of graphene physical and chemical parameters, but also for graphene applications in neuroscience. Moreover, this approach can be translated to other 2D emerging materials and will likely advance the understanding of their toxicology.

Strategies for the Controlled Covalent Double Functionalization of Graphene Oxide.

Graphene oxide (GO) is a versatile platform with unique properties that have found broad applications in the biomedical field. Double functionalization is a key aspect in the design of multifunctional GO with combined imaging, targeting, and therapeutic properties. Compared to noncovalent functionalization, covalent strategies lead to GO conjugates with a higher stability in biological fluids. However, only a few double covalent functionalization approaches have been developed so far. The complexity of GO makes the derivatization of the oxygenated groups difficult to control. The combination of a nucleophilic epoxide ring opening with the derivatization of the hydroxyl groups through esterification or Williamson reaction was investigated. The conditions were selective and mild, thus preserving the structure of GO. Our strategy of double functionalization holds great potential for different applications in which the derivatization of GO with different molecules is needed, especially in the biomedical field.

Kinetics of 1H-13C multiple-contact cross-polarization as a powerful tool to determine the structure and dynamics of complex materials: application to graphene oxide.

Hartmann-Hahn cross-polarization (HHCP) is the most widely used solid-state NMR technique to enhance the magnetization of dilute spins from abundant spins. Furthermore, as the kinetics of CP depends on dipolar interactions, it contains valuable information on molecular structure and dynamics. In this work, analytical solutions are derived for the kinetics of HHCP and multiple-contact CP (MC-CP) using both classical and non-classical spin-coupling models including the effects of molecular dynamics and several 1H, 13C relaxation and 1H-13C CP experiments are performed in graphene oxide (GO). HHCP is found to be inefficient in our GO sample due to very fast 1H T1ρ relaxation. By contrast, the MC-CP technique which alleviates most of the magnetization loss by 1H T1ρ relaxation leads to a much larger polarization transfer efficiency reducing the measuring time by an order of magnitude. A detailed analysis of the HHCP and MC-CP kinetics indicates the existence of at least two different kinds of hydroxyl (C-OH) functional groups in GO, the major fraction (∼90%) of these groups being in the unusual "slow CP regime" in which the rate of 1H T1ρ relaxation is fast compared to the rate of cross-polarization. This 13C signal component is attributed to mobile C-OH groups interacting preferentially with fast-relaxing water molecules while the remaining carbons (∼10%) in the usual "fast CP regime" are assigned to C-OH groups involved in hydrogen bonding with neighboring hydroxyl and/or epoxy groups.