New Insight into the Fluorescence Quenching of Nitrogen-Containing Carbonaceous Quantum Dots-From Surface Chemistry to Biomedical Applications.

Carbon-based quantum dots are widely suggested as fluorescent carriers of drugs, genes or other bioactive molecules. In this work, we thoroughly examine the easy-to-obtain, biocompatible, nitrogen-containing carbonaceous quantum dots (N-CQDs) with stable fluorescent properties that are resistant to wide-range pH changes. Moreover, we explain the mechanism of fluorescence quenching at extreme pH conditions. Our in vitro results indicate that N-CQDs penetrate the cell membrane; however, fluorescence intensity measured inside the cells was lower than expected from carbonaceous dots extracellular concentration decrease. We studied the mechanism of quenching and identified reduced form of ß-nicotinamide adenine dinucleotide (NADH) as one of the intracellular quenchers. We proved it experimentally that the elucidated redox process triggers the efficient reduction of amide functionalities to non-fluorescent amines on carbonaceous dots surface. We determined the 5 nm-wide reactive redox zone around the N-CQD surface. The better understanding of fluorescence quenching will help to accurately quantify and dose the internalized carbonaceous quantum dots for biomedical applications.

A New Approach to Obtaining Nano-Sized Graphene Oxide for Biomedical Applications.

Graphene oxide (GO) is one of the most exciting and widely used materials. A new method of nanographene oxide (n-GO) formation is presented. The described unique sequence of ultrasonication in dimethyl sulfoxide solution allows us to obtain different sizes of n-GO sheets by controlling the timing of the cutting and re-aggregation processes. The obtained n-GO exhibits only minor spectral changes, mainly due to the formation of S-containing surface groups; thus, it can be concluded that the material is not reduced during the process. Maintaining the initial oxygen functionalities together with the required nano-size (down to 200 nm) and high homogeneity are beneficial for extensive applications of n-GO. Moreover, we prove that the obtained material is evidently biocompatible. The calculated half-maximal effective concentration (EC50) increases by 5-fold, i.e., from 50 to 250 µg/mL, when GO is converted to n-GO. As a consequence, the new n-GO neither disturbs blood flow even in the narrowest capillaries nor triggers a toxic influence in surrounding cells. Thus, it can be a serious candidate for drugs and biomolecule carriers administered systemically.

Cytotoxic or Not? Disclosing the Toxic Nature of Carbonaceous Nanomaterials through Nano-Bio Interactions.

The cytotoxic influence of two different carbonaceous nanomaterials on human mesenchymal stem cells (MSCs) cultured in vitro was compared in the short (1-3 days) and long term (up to 60 days). Amorphous carbon and single-walled carbon nanotubes were chosen and evaluated due to their contrasting physicochemical properties. Both materials, though supposed similarly low-toxic in basic short-term cytotoxicity assays, demonstrated dramatically different properties in the long-term study. The surface chemistry and biomolecule-adsorption capacity turned out to be crucial factors influencing cytotoxicity. We proved that amorphous carbon is able to weakly bind a low-affinity protein coat (so-called soft corona), while carbon nanotubes behaved oppositely. Obtained results from zeta-potential and adsorption measurements for both nanomaterials confirmed that a hard protein corona was present on the single-walled carbon-nanotube surface that aggravated their cytotoxic influence. The long-term exposure of the mesenchymal stem cells to carbon nanotubes, coated by the strongly bound proteins, showed a significant decrease in cell-growth rate, followed by cell senescence and death. These results are of great importance in the light of increasing nanomaterial applications in biomedicine and cell-based therapies. Our better understanding of the puzzling cytotoxicity of carbonaceous nanomaterials, reflecting their surface chemistry and interactions, is helpful in adjusting their properties when tailored for specific applications.

Electrophoretic Deposition of Layer-by-Layer Unsheathed Carbon Nanotubes-A Step Towards Steerable Surface Roughness and Wettability.

It is well known that carbon nanotube (CNT) oxidation (usually with concentrated HNO3) is a major step before the electrophoretic deposition (EPD). However, the recent discovery of the "onion effect" proves that multiwalled carbon nanotubes are not only oxidized, but a simultaneous unsheathing process occurs. We present the first report concerning the influence of unsheathing on the properties of the thus-formed CNT surface layer. In our study we examine how the process of gradual oxidation/unsheathing of a series of multiwalled carbon nanotubes (MWCNTs) influences the morphology of the surface formed via EPD. Taking a series of well-characterized and gradually oxidized/unsheathing Nanocyl MWCNTs and performing EPD on a carbon fiber surface, we analyzed the morphology and wettability of the CNT surfaces. Our results show that the water contact angle could be gradually changed in a wide range (125-163°) and the major property determining its value was the diameter of aggregates formed before the deposition process in the solvent. Based on the obtained results we determined the parameters having a crucial influence on the morphology of created layers. Our results shed new light on the deposition mechanism and enable the preparation of surfaces with steerable roughness and wettability.

Carbonaceous Nanomaterials-Mediated Defense Against Oxidative Stress.

The concept of nanoscale materials and their applications in industrial technologies, consumer goods, as well as in novel medical therapies has rapidly escalated in the last several years. Consequently, there is a critical need to understand the mechanisms that drive nanomaterials biocompatibility or toxicity to human cells and tissues. The ability of nanomaterials to initiate cellular pathways resulting in oxidative stress has emerged as a leading hypothesis in nanotoxicology. Nevertheless, there are a few examples revealing another face of nanomaterials - they can alleviate oxidative stress via decreasing the level of reactive oxygen species. The fundamental structural and physicochemical properties of carbonaceous nanomaterials that govern these anti-oxidative effects are discussed in this article. The signaling pathways influenced by these unique nanomaterials, as well as examples of their applications in the biomedical field, e.g. cell culture, cell-based therapies or drug delivery, are presented. We anticipate this emerging knowledge of intrinsic anti-oxidative properties of carbon nanomaterials to facilitate the use of tailored nanoparticles in vivo.

Chemical and Biochemical Approach to Make a Perfect Biocatalytic System on Carbonaceous Matrices.

Enzymatic processes are widely used in food industry, pharmacy, cosmetic and household chemistry, and medicine. However, the common and efficient application of the biological catalysts is limited by a number of factors that influence enzymes activity. One of the most frequent methods to improve the biocatalysts' properties is immobilization. This chapter presents a recent overview of our attempts to obtain the perfect biocatalytic system. The experimental approach, proposed in this chapter, includes the critical points like: the choice of adequate immobilization method, most suitable carrier, determination of enzyme kinetic parameters, stability, and toxicity of obtained systems. As carbon materials including graphene-derived materials offer unique properties and a plenty of different modifications, these parameters seem to be of decisive importance to understand chemistry of complex systems. Consideration of all the mentioned requirements lead us to the conclusion that graphene oxide could be the best candidate for support in perfect biocatalytic systems.

Graphene Oxide-Mediated Protection from Photodamage.

This Letter presents the unique properties of graphene oxide (GO) as a multitask material protecting from UVB-induced photodamage. Three mechanisms of GO action on fibroblast in vitro cultures are verified here: physical - a barrier blocking UV radiation; chemical - antioxidative activity; and biological - activation of cellular antioxidative defense. The changes in GO physicochemical properties appearing due to UVB exposure underpin the observed UV protection phenomena. The results reveal the simultaneous occurrence of two opposed processes, i.e., under small doses of UVB, the tested material undergoes oxidation and sp2 network rebuilding. In the vicinity of the GO surface, the locally triggered high temperature is responsible for a reduction process, while strong oxidative agents such as OH radicals cause parallel GO oxidation. This phenomenon is enabled thanks to the exceptional properties of carbonaceous materials. As a consequence, GO turns out to be a multitask UV protector increasing fibroblast survival.

Novel biocatalytic systems for maintaining the nucleotide balance based on adenylate kinase immobilized on carbon nanostructures.

In this study graphene oxide (GO), carbon quantum dots (CQD) and carbon nanoonions (CNO) have been characterized and applied for the first time as a matrix for recombinant adenylate kinase (AK, EC 2.7.4.3) immobilization. AK is an enzyme fulfilling a key role in metabolic processes. This phosphotransferase catalyzes the interconversion of adenine nucleotides (ATP, ADP and AMP) and thereby participates in nucleotide homeostasis, monitors a cellular energy charge as well as acts as a component of purinergic signaling system. The AK activity in all obtained biocatalytic systems was higher as compared to the free enzyme. We have found that the immobilization on carbon nanostructures increased both activity and stability of AK. Moreover, the biocatalytic systems consisting of AK immobilized on carbon nanostructures can be easily and efficiently lyophilized without risk of desorption or decrease in the catalytic activity of the investigated enzyme. The positive action of AK-GO biocatalytic system in maintaining the nucleotide balance in in vitro cell culture was proved.

Cystine-based MBioF for Maintaining the Antioxidant-Oxidant Balance in Airway Diseases.

Reactive oxygen species, contributing to oxidant-antioxidant imbalance, initiate damage to the airways cells, inflammatory processes, and further pathophysiological effects. Enhancing antioxidant properties is the main prophylactic and therapeutic challenge. In this work, a newly synthesized and biocompatible structure of the metal-biomolecule frameworks (MBioF) harnessing cystine as a linker and magnesium as metal nodes is presented. This structure provides crucial sulfhydryl groups of cysteine, with antioxidant activity, released stepwise in the site of delivery. We prove that once released, the compounds of MBioF increase the intracellular level of cysteine and total antioxidative capability of airway cells. Presented MBioF structures offer new perspectives for clinical applications as therapeutics or preventatives maintaining the antioxidant-oxidant balance.

Controlling enzymatic activity by immobilization on graphene oxide.

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Air pollution, UV irradiation and skin carcinogenesis: what we know, where we stand and what is likely to happen in the future?

The link between air pollution, UV irradiation and skin carcinogenesis has been demonstrated within a large number of epidemiological studies. Many have shown the detrimental effect that UV irradiation can have on human health as well as the long-term damage which can result from air pollution, the European ESCAPE project being a notable example. In total, at present around 2800 different chemical substances are systematically released into the air. This paper looks at the hazardous impact of air pollution and UV and discusses: 1) what we know; 2) where we stand; and 3) what is likely to happen in the future. Thereafter, we will argue that there is still insufficient evidence of how great direct air pollution and UV irradiation are as factors in the development of skin carcinogenesis. However, future prospects of progress are bright due to a number of encouraging diagnostic and preventive projects in progress at the moment.

Conscious Changes of Carbon Nanotubes Cytotoxicity by Manipulation with Selected Nanofactors.

We discuss eight major challenges in the field of carbon nanomaterial toxicity. Generally, we pick up some of them, and the most important challenge is searching of the qualitative relationships between nanofactors and cytotoxicity. This is important since it can provide the possibility of conscious changes of carbon nanotubes cytotoxicity by manipulation with selected nanofactors. Therefore, the toxicity of a series of gradually oxidized carbon nanotubes is studied. We show, for the first time, that toxicity of those materials depends strongly on the ratio of acidic to basic group concentration--the higher is this ratio value, the more toxic are nanotubes. In this way, by changing this ratio, one can change toxicity. This correlation is more evident after ultrasonication, and it is connected with the accessibility of charged groups for interactions with proteins. Toxicity also depends on the ability of nanotubes for protein adsorption. We suggest that the changes in the protein composition of medium, especially lack of important growth factors, inhibit cell proliferation.