Protein Corona Hinders N-CQDs Oxidative Potential and Favors Their Application as Nanobiocatalytic System.

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.

Porphyrin Based 2D-MOF Structures as Dual-Kinetic Sorafenib Nanocarriers for Hepatoma Treatment.

The existing clinical protocols of hepatoma treatment require improvement of drug efficacy that can be achieved by harnessing nanomedicine. Porphyrin-based, paddle-wheel framework (PPF) structures were obtained and tested as dual-kinetic Sorafenib (SOR) nanocarriers against hepatoma. We experimentally proved that sloughing of PPF structures combined with gradual dissolving are effective mechanisms for releasing the drug from the nanocarrier. By controlling the PPF degradation and size of adsorbed SOR deposits, we were able to augment SOR anticancer effects, both in vitro and in vivo, due to the dual kinetic behavior of SOR@PPF. Obtained drug delivery systems with slow and fast release of SOR influenced effectively, although in a different way, the cancer cells proliferation (reflected with EC50 and ERK 1/2 phosphorylation level). The in vivo studies proved that fast-released SOR@PPF reduces the tumor size considerably, while the slow-released SOR@PPF much better prevents from lymph nodes involvement and distant metastases.

Chemical Characteristics and Antioxidant Activity of Arctostaphylos uva-ursi L. Spreng. at the Southern Border of the Geographical Range of the Species in Europe.

The bearberry (Arctostaphylos uva-ursi L. Spreng.) is a source of herbal material-bearberry leaf (Uvae ursi folium), which is highly valued and sought by pharmaceutical and cosmetic industries. For many years, leaves of this plant have been used in traditional medicine as a diuretic, antimicrobial, and anti-inflammatory agent for various diseases of the urogenital tract. The bearberry has also been proposed as a natural antioxidant additive due to the high contents of phenolic compounds in its leaves. The study was focused on characterization of the basic phytochemical composition and antioxidant activity of extracts derived from bearberry leaves collected from plants located at the southern border of the geographical range of the species in Europe. The investigated herbal material is characterized by a different chemical profile compared to the chemical profiles of bearberry found in other parts of the continent. Bearberry extracts from plants growing in two different habitat types-heathlands and pine forests showed a wide range of variation, especially in the concentration of hyperoside, corilagin, and methylartutin and the total flavonoid contents. In addition to arbutin, bearberry can be a valuable source of phenolic compounds, which are mainly responsible for the antioxidant properties of extracts. The high content of phenols and high values of antioxidant parameters indicate a high potential of bearberry leaves to be used as a powerful natural source of antioxidants in herbal preparations. Therefore, the A. uva-ursi populations can be a source of plant material for pharmaceutical, cosmetic, and food industries.

Synthesis, characterization and evaluation of antimicrobial and cytotoxic activities of biogenic silver nanoparticles synthesized from Streptomyces xinghaiensis OF1 strain.

We report synthesis of silver nanoparticles (AgNPs) from Streptomyces xinghaiensis OF1 strain, which were characterised by UV-Vis and Fourier transform infrared spectroscopy, Zeta sizer, Nano tracking analyser, and Transmission electron microscopy. The antimicrobial activity of AgNPs alone, and in combination with antibiotics was evaluated against bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis, and yeasts viz., Candida albicans and Malassezia furfur by using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum biocidal concentration of AgNPs against bacterial and yeast strains were determined. Synergistic effect of AgNPs in combination with antibacterial and antifungal antibiotics was determined by FIC index. In addition, MTT assay was performed to study cytotoxicity of AgNPs alone and in combination with antibiotics against mouse fibroblasts and HeLa cell line. Biogenic AgNPs were stable, spherical, small, polydispersed and capped with organic compounds. The variable antimicrobial activity of AgNPs was observed against tested bacteria and yeasts. The lowest MIC (16 µg ml-1) of AgNPs was found against P. aeruginosa, followed by C. albicans and M. furfur (both 32 µg ml-1), B. subtilis and E. coli (both 64 µg ml-1), and then S. aureus and Klebsiella pneumoniae (256 µg ml-1). The high synergistic effect of antibiotics in combination with AgNPs against tested strains was found. The in vitro cytotoxicity of AgNPs against mouse fibroblasts and cancer HeLa cell lines revealed a dose dependent potential. The IC50 value of AgNPs was found in concentrations of 4 and 3.8 µg ml-1, respectively. Combination of AgNPs and antibiotics significantly decreased concentrations of both antimicrobials used and retained their high antibacterial and antifungal activity. The synthesis of AgNPs using S. xinghaiensis OF1 strain is an eco-friendly, cheap and nontoxic method. The antimicrobial activity of AgNPs could result from their small size. Remarkable synergistic effect of antibiotics and AgNPs offer their valuable potential in nanomedicine for clinical application as a combined therapy in the future.

Neurogenic Differentiation of Mesenchymal Stem Cells Induces Alterations in Extracellular Nucleotides Metabolism.

The presented results show for the first time that the neurogenic transdifferentiation of hUC-MSCs considerably changes the elements of purinergic signaling profile. Although, it has been demonstrated in the literature that extracellular nucleotides and nucleosides determine the fate of mesenchymal and neural stem cells, there is lack of comprehensive studies on the activity of ecto-enzymes metabolizing nucleotides on the surface of neurogenically induced cells. Our study shows that human UC-MSCs sense the microenvironment and adjust their response to the environmental signals for example, adenine nucleotides and nucleosides. Nucleotides, and not adenosine, signaling alters the biological potential of MSCs-decreases their proliferation rate, increases the neurogenic transdifferentiation efficiency expressed as the number of positively labeled NCAM+ and A2B5+ cells and simultaneously increases the ecto-nucleotidases activity on neural- and glial-committed precursors. Purines implication in the proliferative and neurogenic potential of hUC-MSCs is of strong importance for the in vitro propagation of hUC-MSCs and for their successive therapeutic applications. J. Cell. Biochem. 118: 478-486, 2017. © 2016 Wiley Periodicals, Inc.

Canine Adipose-Derived Stem Cells: Purinergic Characterization and Neurogenic Potential for Therapeutic Applications.

The presented results evidence that canine adipose-derived stem cells (ADSCs) represent the premature population of stem cells with great biological potential and properties. ADCS are easy to obtain and culture, able to differentiate into the neurogenic lineage as well as it is easy to control their proliferation rate with nucleotides and nucleosides or analogues. We report that in vitro cultured canine ADSCs response to adenosine- and ATP-mediated stimulation. Differences in canine ADSCs and human mesenchymal stem cells in ecto-nucleotidase activity have been observed. The ecto-nucleotidase activity changes during ADSCs in vitro transdifferentiation into neurogenic lineage are fast and simple to analyze. Therefore, the simple analysis of ecto-enzymes activity allows for verification of the stem cells quality: their stemness or initiation of the differentiation process. The biological potential of the cells isolated from canine fat, as well as the good quality control of this cell culture, make them a promising tool for both experimental and therapeutic usage. J. Cell. Biochem. 118: 58-65, 2017. © 2016 Wiley Periodicals, Inc.

High Quality Independent From a Donor: Human Amniotic Fluid Derived Stem Cells-A Practical Analysis Based on 165 Clinical Cases.

The aim of the study was to extend the potential use of human stem cells isolated from amniotic fluid in medical applications by confirming their high homogeneity and quality. Amniotic fluid samples were collected during amniocentesis from 165 women during pregnancy. The proliferation rate, clonogenicity, karyotype, aging process, pluripotent cell markers, expression of surface markers, and the potential to differentiate into adipose, bone and cartilage cells of hAFSCs were analyzed. Obtained results revealed that mesenchymal stem cells could be derived successfully from amniotic fluid, which exhibit properties comparable with MSCs of other origins. It is the first study, in which such a large group of patients was involved. Comprehensive statistical and biological analysis were conducted some of which clearly being innovative in relation to human amniotic fluid-derived stem cells. J. Cell. Biochem. 118: 116-126, 2017. © 2016 Wiley Periodicals, Inc.

Silver nanoparticles from Pilimelia columellifera subsp. pallida SL19 strain demonstrated antifungal activity against fungi causing superficial mycoses.

In this study, we present the in vitro antifungal activity of silver nanoparticles (AgNPs) synthesized from acidophilic actinobacterium Pilimelia columellifera subsp. pallida SL19 strain, alone and in combination with antibiotics viz., amphotericin B, fluconazole, and ketoconazole against pathogenic fungi, namely Candida albicans, Malassezia furfur, and Trichophyton erinacei. The minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) of AgNPs against test fungi were evaluated. The fractional inhibitory concentration (FIC) index was determined to estimate antifungal activity of AgNPs combined with antibiotics. Antifungal activity of AgNPs varied among the tested fungal strains. M. furfur was found to be most sensitive to biogenic silver nanoparticles, followed by C. albicans and T. erinacei. The lowest MIC of AgNPs was noticed against M. furfur (16 µg ml-1 ). Synergistic effect was observed on C. albicans when AgNP were combined with amphotericin B and ketoconazole and on M. furfur with fluconazole and ketoconazole (FIC index of 0.5). Cytotoxic effect of AgNPs on HeLa and 3T3 cell lines was evaluated. The IC50 values were found to be 55 and 25 µg ml-1 , respectively. The present study indicates that silver nanoparticles from P. columellifera subsp. pallida SL19 strain have antifungal activity, both alone and in combination with antibiotics, and offer a valuable contribution to nanomedicine.

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.

Chondrogenic Differentiation of Human Mesenchymal Stem Cells Results in Substantial Changes of Ecto-Nucleotides Metabolism.

Mesenchymal stem cells (MSCs) are population of adult stem cells and attractive candidates for cartilage repair due to their chondrogenic potential. Purinergic compounds (purinergic receptors and ecto-enzymes metabolizing nucleotides), together with nucleotides/nucleosides present in the extracellular environment, are known to play a key role in controlling the stem cells biological potential to proliferate and differentiate. Despite the available literature pointing to the importance of purinergic signaling in controlling the fate of MSCs, the research results linking nucleotides and ecto-nucleotidases with MSCs chondrogenic differentiation are indigent. Therefore, the aim of presented study was the characterization of the ecto-nucleotides hydrolysis profile and ecto-enzymes expression in human umbilical cord-derived MSCs and chondrogenically induced MSCs. We described substantial changes of ecto-nucleotides metabolism and ecto-enzymes expression profiles resulting from chondrogenic differentiation of human umbilical cord-derived MSCs. The increased rate of ADP hydrolysis, measured by ecto-nucleotidases activity, plays a pivotal role in the regulation of cartilage formation and resorption. Despite the increased level of NTPDase1 and NTPDase3 mRNA expression in chondrogenically induced MSCs, their activity toward ATP remains quite low. Supported by the literature data, we hypothesize that structure-function relationships in chondrogenic lineage dictate the direction of nucleotides metabolism. In early neocartilage tissue, the beneficial role of ATP in improving biomechanical properties of cartilage does not necessitate the high rate of enzymatic ATP degradation.

The role of purinergic signaling in the etiology of migraine and novel antimigraine treatment.

Etiopathogenesis of migraine involves different structures of the central nervous system: the trigeminal nerve with nuclei located in the brain stem, vascular system, and the cerebral cortex as well as diverse mechanisms and pathological processes. The multidirectional action of purines in different cell types (blood vessels, neurons, and satellite glial cells) and through different types of purinergic receptors contributes to the etiopathogenesis of migraine pain. Adenosine triphosphate (ATP) and its derivatives are involved in initiation and propagation of migrenogenic signals in several ways: they participate in vasomotor mechanism, cortical spreading depression, and in fast transmission or cross-excitation based on the satellite glial cells in trigeminal ganglion. Contribution of purinergic signaling in the conduction of pain is realized through the activation of P1 and P2 receptors expressed widely in the central nervous system: on the neurons and glial cells as well as on the smooth muscles and endothelium in the vascular system. Therefore, the purinergic receptors can be an excellent target for pharmacologists constructing new antimigraine therapeutics. Moreover, the mechanisms facilitating ATP and adenosine degradation may prevent vasodilatation and thus avoid a secondary central sensitization during a migraine attack. Thus, agonists and antagonists of P receptors as well as ecto-enzymes metabolizing nucleotides/nucleosides could gain the growing attention as therapeutic agents.

[Human adenylate kinases - classification, structure, physiological and pathological importance]. / Kinazy adenylanowe czlowieka-klasyfikacja, budowa oraz znaczenie w fizjologii i patologii.

Adenylate kinase (AK, EC 2.7.4.3) is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy ß - and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd) and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD)) link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.

[Stem cells and senescence]. / Komórki macierzyste a starzenie.

Stem cells are undifferentiated cells that can differentiate into specialized cells, that build the whole body. These rare cells are required for homeostasis and tissue replacement throughout the human lifespan, and appear to be characterized by a few specific physiological and biochemical properties, particularly the capacity for self-renewal. Recent studies suggest that stem cells may undergo senescence, what plays a crucial role in organismal aging. Importantly, both senescence and apoptosis are anti-cancer mechanisms that counteract neoplastic transformation of stem cells. On the other hand, mechanisms that suppress the development of cancer may also induce an unwanted consequence: a decline in the number and functional alterations of stem cells with advancing age. These functional changes reflect harmful effects of age on the genome, epigenome, and proteome of stem cells. Some of which arise cell independently and others which are imposed by an age-related change in the local milieu or systemic environment. Remarkably, some of the changes, particularly epigenomic and proteomic ones, are potentially reversible, and both environmental (e.g. caloric restrictions, hypoxia) and genetic interventions can lead to inducible pluripotency. Here, we discuss recent discoveries in the field of senescence of stem cells. These findings have profound implications, not only for our understanding of stem cells' biology and organismal aging, but also for stem cell-based regenerative medicine and stem cell-based therapy of age-related diseases.

[Three-dimensional cell cultures. Applications in basic science and biotechnology]. / Trójwymiarowe hodowle komórek--zastosowania w badaniach podstawowych i inzynierii tkankowej.

The tissue culture technique is widely used in biochemical and molecular studies, offering accessibility to biological material, high reproducibility of results and high throughput format, comparing with organ cultures. However, traditional, two-dimensional cultures (2D cultures) poorly represent the microenvironment of a tissue, and they are gradually replaced with 3D cultures, that enable formation of intercellular contacts, signaling pathways and gene expression characteristic for tissue in vivo. This paper presents the biology of three-dimensional cultures (spheroids), their applications in basic science and biotechnology and methods of spheroids formation.

Purinergic approach to effective glioma treatment with temozolomide reveals enhanced anti-cancer effects mediated by P2X7 receptor.

The purinergic signaling pathway is the oldest evolutionary transmitter system that regulates a wide array of physiological and pathophysiological processes in central nervous system. However, the question of how the purinergic compounds interact with administrated drugs is rarely addressed. We aimed to clarify the interplay between purinergic signaling and chemotherapeutic drug temozolomide (TMZ) in human glioma cell line. We applied an initial retinoic acid-induced differentiation of A172 glioma cells and tested the P2X7 receptor expression in undifferentiated and differentiated gliomas. We compared the P2X7 receptor agonists/antagonists influence and their co-action with TMZ in both cell types through assessment of cell proliferation, viability and migrative properties. Molecular docking allowed to indicate the potential binding site for TMZ in the structure of hP2X7 receptor. Differentiated cells turned out to be more susceptible to ATP and TMZ alone but also to the concerted action of TMZ and ATP. Enhanced effects triggered by ATP and TMZ treatment include the decreased by 70% viability, and reduced migration ability of differentiated A172 glioma cells. Noteworthy, these results can be achieved already at low non-toxic ATP concentration and at reduced to 125 µM effective concentration of TMZ. Therefore, ATP molecules must be present and maintained at appropriate concentration in glioma cells microenvironment to achieve their co-action with TMZ and enhanced anti-cancer activity. All that, in turn, could shorten the therapy, increase its efficacy and limit the side effects for the patient. Our purinergic approach creates a promising perspective for developing novel combined oncological therapies.

Adenylate kinase immobilized on graphene oxide impairs progression of human lung carcinoma epithelial cells through adenosinergic pathway.

Purinergic signaling, the oldest evolutionary transmitter system, has been increasingly studied as a pivotal target for novel anti-cancer therapies. In the present work, the developed nanobiocatalytic system consisting of adenylate kinase immobilized on graphene oxide (AK-GO) was characterized in terms of its physicochemical and biochemical properties. We put special emphasis on the AK-GO influence on purinergic signaling components, that is, ecto-nucleotides concentration and ecto-enzymes expression and activity in human lung carcinoma epithelial (A549) cells. The immobilization-dependent modification of AK kinetic parameters allowed for the removal of ATP excess while maintaining low ATP concentrations, efficient decrease in adenosine concentration, and control of the nucleotide balance in carcinoma cells. The cyto- and hemocompatibility of developed AK-GO nanobiocatalytic system indicates that it can be successfully harnessed for biomedical applications. In A549 cells treated with AK-GO nanobiocatalytic system, the significantly decreased adenosinergic signaling results in reduction of the proliferation and migration capability of cancer cells. This finding is particularly relevant in regard to AK-GO prospective anti-cancer applications.

Nitrogen plasma modification boosts up the hemocompatibility of new PVDF-carbon nanohorns composite materials with potential cardiological and circulatory system implants application.

To design new material for blood-related applications one needs to consider various factors such as cytotoxicity, platelet adhesion, or anti-thrombogenic properties. The aim of this work is the design of new, highly effective materials possessing high blood compatibility. To do this, the new composites based on the poly(vinylidene fluoride) (PVDF) support covered with a single-walled carbon nanohorns (CNHs) layer were prepared. The PVDF-CNHs composites were subsequently used for the first time in the hemocompatibility studies. To raise the hemocompatibility a new, never applied before for CNHs, plasma-surface modifications in air, nitrogen and ammonia were implemented. This relatively cheap, facile and easy method allows generating the new hybrid materials with high effectiveness and significant differences in surface properties (water contact angle, surface ζ-potential, and surface functional groups composition). Changing those properties made it possible to select the most promising samples for blood-related applications. This was done in a fully controlled way by applying Taguchi's "orthogonal array" procedure. It is shown for the first time that nitrogen plasma treatment of new surfaces is the best tool for hemocompatibility rise and leads to very low blood platelet adhesion, no cytotoxicity, and excellent performance in thromboelastometry and hemolysis tests. We propose a possible mechanism explaining this behavior. The optimisation results are coherent with biological characterisation and are supported with Hansen Solubility Parameters. New surfaces can find potential applications in cardiological and circulatory system implants as well as other blood-related biomaterials.

[The role of ectonucleotides metabolizing enzymes in purinergic signaling]. / Rola enzymów metabolizujacych ektonukleotydy w sygnalizacji z udzialem puryn.

Purinergic signaling plays an important role in the regulation of many physiological processes. The concentration of nucleotides in extracellular space is controlled by at least two families of nucleotidases: NPPases and NTPDases. These families are examples of convergent evolution of proteins. Above ezymes are not phylogenetically related, but they catalyze the same type of reaction. They hydrolyzed tri- and diphosphonucleosides to monophosphonucleosides and orthophosphate or pyrophosphate. This degradation terminates the nucleotide signaling process and also produces other signaling molecules like ADP, and with 5'-nucleotidase, adenosine. Most of known animal NPPases and NTPDases were found as membranous ectoenzymes or soluble proteins localized in tissue fluids. The aim of this work is to provide information about localization, structure, properties and function of NPPases and NTPDases in the regulation of extracellular concentration of nucleotides and purinergic signaling.

Nitric-Acid Oxidized Single-Walled Carbon Nanohorns as a Potential Material for Bio-Applications-Toxicity and Hemocompatibility Studies.

The results of in vitro studies of single-walled carbon nanohorn (SWCNH) oxidized materials' cytotoxicity obtained by the cell membrane integrity (Neutral Red Uptake (NRU)) and metabolic activity (by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) on A549 and human dermal fibroblasts (HDF) cell lines are presented. We also present hemocompatibility studies on human and porcine blood, and an erythrocyte concentrate to prove that the obtained samples will not interfere with blood components. Characterization of the materials is supplemented by ζ-potential measurements, Transmission Electron Microscope (TEM) imaging, and thermogravimetric studies (TG). The presented results show the correlation between the specific surface area of materials and the platelet aggregation, when the ID/IG ratio determined from Raman spectra correlates with hemoglobin release from the erythrocytes (in whole blood testing). A plausible mechanism explaining the observed correlations is given. The cytotoxicity and hemocompatibility studies prove that the studied materials are acceptable for use in biomedical applications, especially a sample SWCNH-ox-1.5 with the best application potential.

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.