N,S-Codoped Carbon Dots with Red Fluorescence and Their Cellular Imaging.

The emergence of carbon dots (C-dots) has aroused increasing attention owing to their excellent chemical and physical properties, such as favorable biocompatibility and an outstanding fluorescence (FL) property. Most reported C-dots show blue emission, which hinders their applications in the biomedical field due to the strong FL background of biosamples. Therefore, strategies for the achievement of long-wavelength fluorescent C-dots are urgently needed. Herein, red emissive biocompatible C-dots were prepared from polythiophene (PTh) through chemical cutting by nitric acid. Various methods were utilized to shed light on the luminescence mechanism of the C-dots. The results demonstrated that FL emission of the prepared C-dots was mainly dominated by sp2 domains. The C-dots were well-applied in in vitro imaging. This work prepared red fluorescent C-dots from the perspective of the structure of C-dots, which would benefit the regulation of the FL of C-dots.

Chitosan quaternary ammonium salt induced mitochondrial membrane permeability transition pore opening study in a spectroscopic perspective.

Chitosan is non-toxic, biodegradable and biocompatible. However, it is insoluble in water, which limits its applications in biomedical areas. Hydroxypropyltrimethyl ammonium chloride chitosan (HACC), a chitosan derivative, can be dissolved in physiological condition and has been widely used in the field of biomedicine and bioengineering. The biological effect of HACC has been extensively studied. However, it is rarely investigated at the subcellular level. To study the biological effect of HACC, mitochondria, energy-producing organelles in eukaryotes, were chosen as a model. The investigation mainly focused on the changes of mitochondrial membrane property in the presence of HACC. Results showed that HACC can induce the collapse of mitochondrial transmembrane potential (∆Ψm), the increase in mitochondrial membrane swelling and the decrease of mitochondrial membrane fluidity, demonstrating that mitochondrial membrane permeability transition pore (mPTP) opening happened. Possible mechanism of mPTP opening investigation indicated that it was occurred in a typical model. In addition, HACC can induce the release of cytochrome C (Cyt c) and affect the respiratory activity of mitochondria. The study will provide a lot of important information on biosafety evaluation of HACC.

Molecular Mechanisms of the Ultra-Strong Inhibition Effect of Oxidized Carbon Dots on Human Insulin Fibrillation.

Amyloid fibrillation of protein is associated with a great variety of pathologic conditions. The aggregation of protein is a complicated process with multisteps, whereas most of the inhibitors with elaborately designed structures can show an inhibition effect only on the nucleation stages of protein fibrillation. Herein, oxidized carbon dots (CDs) were achieved to study the relationship between the surface properties of CDs and their inhibition effect on human insulin (HI) fibrillation. More oxygen-containing function groups can be obtained after oxidation reaction of CDs, such as -OH and -COOH. The results show that 10-1 CDs (the mass ratios of CD/KMnO4 is 10:1), with the highest carboxyl group content, possess the best inhibition ability. All the nucleation, growth, and final phases can be retarded by 10-1 CDs, which have been studied in detail by fluorescence spectra. However, CDs without oxidation can show only a weak inhibition effect on the nucleation stage. The 10-1 CDs is demonstrated to binding with HI monomers much stronger than that of CDs by isothermal titration calorimetry (ITC). Moreover, molecular dynamics simulations (MD) studies indicate that CDs with more carboxyl groups can show stronger affinities with native or unfolded HI monomers, which may be mainly derived from the active binding sites of histidine residues (His5 and His10) on B-chain through electrostatic interaction. Because the unfolding of B-chain in HI is prior to that of A-chain in our MD simulations, the later aggregation of HI can be inhibited effectively by the stronger binding forces between 10 and 1 CDs and the B-chain of HI.

Single-step synthesis of highly photoluminescent carbon dots for rapid detection of Hg2+ with excellent sensitivity.

Carbon dots (C-dots) are superior in the aspects of excellent water solubility, good biocompatibility, environmentally friendliness and non-blinking fluorescence. In this work, highly photoluminescent small-size C-dots (QY = 18.8%, quinine sulfate as standard) with narrow size distribution (1.70 ±â€¯0.21 nm) have been synthesized by using citric acid and triethylamine through hydrothermal method. The optimal excitation and emission wavelength of C-dots are 350 nm and 437 nm, respectively. And the prepared C-dots display excitation-independent behavior due to less surface defects and uniform size. Interestingly, the fluorescence of C-dots could be rapidly and selectively quenched by Hg2+ within 200 s at room temperature without further modification. Under optimal conditions, the limit of detection (LOD) was measured to be nanomolar level (2.8 nM) with a linear range of 0.05-7 µM, lower than the previous published reports. Furthermore, our results reveal that static quenching mechanism was dominated in the process in which Hg2+ coordinate with the oxygen-containing groups of C-dots to form nonfluorescent complexes. And only the addition of Hg2+ destroyed the surface defects of C-dots resulting in the fluorescent quenching. The presence of other common interfering metal ions reported in previous literature (Ag+, Cu2+, Fe3+) do not affect the surface defects, which has rarely been reported before. Besides, this sensing platform has been further successfully applied to the label free detection of Hg2+ in tap water and living cells. These conclusions demonstrate the great potential of our C-dots in selective detection of environmental and cellular Hg2+, which may achieve a lot of achievements in clinical diagnosis and other biological researches.

Fluorescent protein nanoparticles: Synthesis and recognition of cellular oxidation damage.

Intracellular reactive oxygen species (ROS) generation are associated with many diseases. Lots of studies focus on the detection of intracellular ROS by small fluorescent molecules. However, ROS recognized by biocompatible nanoparticles are relatively less reported. It is widely known that albumin-based nanomaterials possess unique advantages in biomedical applications because they are biodegradable and biocompatible. Herein, fluorescent protein nanoparticles (PNPs) were prepared using BSA as a starting material without introducing extra fluorescent molecules. The blue fluorescent PNPs were well characterized by FL, FTIR, CD, TEM, DLS, etc. It was revealed that the PNPs exhibited two types of emissive centers through FL spectra and the fluorescence lifetimes. Further mechanism study indicated that the fluorescence of the PNPs was mainly derived from three kinds of aromatic amino acids, namely tryptophan, tyrosine and phenylalanine. Moreover, the fluorescence properties of the PNPs were tightly related to pH. The PNPs displayed excellent stabilities under harsh conditions as well as physiological conditions. In addition, the PNPs (200 µg/mL) were nontoxic to HeLa and GES-1 cell lines, showing good biocompatibility. The cellular uptake of PNPs was occurred only when the cells were stressed with glucose oxidase or H2O2, thereafter the bright blue fluorescence was observed, indicating that it could be utilized for the recognition of cellular oxidation damage. These findings will offer novel clues for the future synthesis of even brighter protein nanoparticles and their biomedical applications.

Ultrasmall silver nanoclusters: Highly efficient antibacterial activity and their mechanisms.

Silver materials have been widely used as antimicrobial agents. Notably, silver nanoparticles have emerged as a new generation of nanoproducts for biomedical and environmental applications in recent years. However, ultrasmall silver nanoclusters (NCs) (∼2 nm) have rarely been used to kill bacteria and their antibacterial mechanisms have not yet been fully elucidated. Herein, we studied the antibacterial activities of bifunctional fluorescent DHLA-AgNCs against three types of bacteria. The results showed that DHLA-AgNCs exhibited excellent antibacterial activities against Gram-negative E. coli, which could efficiently inhibit the growth of E. coli DH 5α and E. coli DSM 4230 cells at a concentration of 15 and 10 µg mL-1, respectively. Meanwhile AgNCs demonstrated no apparent antibacterial activity against Gram-positive S. aureus. Then, the antibacterial mechanisms of AgNCs were systematically investigated. We found that AgNCs affected the growth of different E. coli strains in different ways. AgNCs inhibited the growth of E. coli DH 5α mainly through damaging the outer cellular membrane and permeating into the cells, followed by the antibacterial effect of the internalized AgNCs and released silver ions. AgNCs, however, inhibited the growth of E. coli DSM 4230 cells mainly through diffusing into E. coli DSM 4230 cells and damaging their respiratory chain. These results clearly indicated that different bacterial strains (e.g. different E. coli strains) should be taken into consideration in future studies. Our work facilitates further investigation of the design of new antibacterial silver nanomaterials with different sizes.

Mitochondrial dysfunction induced by ultra-small silver nanoclusters with a distinct toxic mechanism.

As noble metal nanoclusters (NCs) are widely employed in nanotechnology, their potential threats to human and environment are relatively less understood. Herein, the biological effects of ultra-small silver NCs coated by bovine serum albumin (BSA) (Ag-BSA NCs) on isolated rat liver mitochondria were investigated by testing mitochondrial swelling, membrane permeability, ROS generation, lipid peroxidation and respiration. It was found that Ag-BSA NCs induced mitochondrial dysfunction via synergistic effects of two different ways: (1) inducing mitochondrial membrane permeability transition (MPT) by interacting with the phospholipid bilayer of the mitochondrial membrane (not with specific MPT pore proteins); (2) damaging mitochondrial respiration by the generation of reactive oxygen species (ROS). As far as we know, this is the first report on the biological effects of ultra-small size nanoparticles (∼2 nm) at the sub-cellular level, which provides significant insights into the potential risks brought by the applications of NCs. It would inspire us to evaluate the potential threats of nanomaterials more comprehensively, even though they showed no obvious toxicity to cells or in vivo animal models. Noteworthy, a distinct toxic mechanism to mitochondria caused by Ag-BSA NCs was proposed and elucidated.

Interactions between carbon nanodots with human serum albumin and γ-globulins: The effects on the transportation function.

Carbon nanodots (C-dots) have attracted great attention as a new class of luminescent nanomaterials due to their superior physical and chemical properties. In order to better understand the basic behavior of C-dots in biological systems, a series of photophysical measurements were applied to study the interactions of C-dots with human serum albumin (HSA) and γ-globulins. The fluorescence of proteins was quenched by the dynamic mechanism rather than the formation of a protein/C-dots complex. The apparent dissociation constants of the C-dots bound to HSA and γ-globulins were of the same order of magnitude. Furthermore, it is proven that C-dots showed little influence on the conformation of HSA and γ-globulins. In addition, Fourier transform infrared and fluorescence spectroscopic studies demonstrated that the interaction between C-dots and two kinds of serum proteins was driven by hydrophobic and van der waals forces. Since the bioavailability of drugs can be modulated by their interactions with proteins, the variations of binding constants of three drugs with HSA and γ-globulins in the presence of different concentrations of C-dots (0-84 µmol L(-1)) have also been analyzed in this work, to reflect the effect of C-dots on the transportation function of HSA and γ-globulins.

Highly Photoluminescent Nitrogen-Doped Carbon Nanodots and Their Protective Effects against Oxidative Stress on Cells.

Highly photoluminescent (PL) (quantum yield = 54%) nitrogen doped carbon nanodots (C-dots) have been prepared through one-step carbonizing citric acid and tris(hydroxymethyl)aminomethane and using oleic acid as solvent. The synthesized C-dots are monodisperse with narrow size distribution (average 1.7 nm). The PL properties of C-dots are pH dependent, and hence, using C-dots as sophisticated pH sensor to detect pH values between 7 and 9 can be expected. In addition, the PL intensity of C-dots remains stable under high ionic strength. The C-dots can protect cells from oxidative stress, which shows potential to expand the biological application of C-dots, especially in medical treatment. The protective mechanism is associated with intracellular reactive oxygen species elimination and the intracellular superoxide dismutase production.

Necrotic cell death induced by the protein-mediated intercellular uptake of CdTe quantum dots.

The toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with four different ligands (MPA, NAC, GSH and dBSA) to HEK293 and HeLa cells were investigated using flow cytometry, spectroscopic and microscopic methods. The results showed that the cytotoxicity of QDs increased in a dose- and time-dependent manner. No appreciable fraction of cells with sub-G1 DNA content, the loss of membrane integrity, and the swelling of nuclei clearly indicated that CdTe QDs could lead to necrotic cell death in HEK293 cells. JC-1 staining and TEM images confirmed that QDs induced MPT, which resulted in mitochondrial swelling, collapse of the membrane potential. MPT is an important step in QDs-induced necrosis. Moreover, QDs induced MPT through the elevation of ROS. The fluorimetric assay and theoretical analysis demonstrated ROS production has been associated with the internalization of QDs with cells. Due to large surface/volume ratios of QDs, when QDs added in the culture medium, serum proteins in the culture medium will be adsorbed on the surface of QDs. This adsorption of serum protein will change the surface properties and size, and then mediate the cellular uptake of QDs via the clathrin-mediated endocytic pathway. After entering into cells, the translocation of QDs in cells is usually via endosomal or lysosomal vesicles. The rapid degradation of QDs in lysosome and the lysosomal destabilization induce cell necrosis. This study provides a basis for understanding the cytotoxicity mechanism of CdTe QDs, and valuable information for safe use of QDs in the future.

Mechanistic studies on the reversible photophysical properties of carbon nanodots at different pH.

The pH-dependent photoluminescence (PL) behavior of carbon nanodots (C-dots) and its mechanism has been exhaustively studied in this work. The PL and UV-vis absorption spectra are reversible in the pH between 3 and 13. We speculate that two kinds of reactions (fast and slow) occurring at the surface of C-dots may contribute to this pH-dependent PL behavior. When C-dots solutions are switched to acidic conditions, they will quickly self-assembled aggregate into larger particles and surface oxygen-related groups of C-dots would be slowly oxidized at room temperature. Moreover, it should be noted that this is the first direct observation of self-assembled aggregation of C-dots under acidic conditions. In addition, the optimal PL spectra of C-dots blue-shift while their sizes increase, so-called 'inverse PL shift' phenomenon is also observed. Meanwhile, as the solution is adjusted to alkaline conditions, a structural tautomerization of C-dots rapidly takes place and hydrogenation/deoxygenation reaction proceeds in a much slower rate. Furthermore, through distinct decay dynamics as well as the characterizations of C-dots at different pHs, the PL properties are proposed to be mainly related to the surface states of C-dots.

Spectroscopic and Microscopic Studies on the Mechanism of Mitochondrial Toxicity Induced by CdTe QDs Modified with Different Ligands.

Quantum dots (QDs) are increasingly applied in sensing, drug delivery, biomedical imaging, electronics industries, etc. Consequently, it is urgently required to examine their potential threat to humans and the environment. In the present work, the toxicity of CdTe QDs with nearly identical maximum emission wavelength but modified with two different ligands (MPA and BSA) to mitochondria was investigated using flow cytometry, spectroscopic, and microscopic methods. The results showed that QDs induced mitochondrial permeability transition (MPT), which resulted in mitochondrial swelling, collapse of the membrane potential, inner membrane permeability to H(+) and K(+), the increase of membrane fluidity, depression of respiration, alterations of ultrastructure, and the release of cytochrome c. Furthermore, the protective effects of CsA and EDTA confirmed QDs might be able to induce MPT via a Ca(2+)-dependent domain. However, the difference between the influence of CdTe QDs and that of Cd(2+) on mitochondrial membrane fluidity indicated the release of Cd(2+) was not the sole reason that QDs induced mitochondrial dysfunction, which might be related to the nanoscale effect of QDs. Compared with MPA-CdTe QDs, BSA-CdTe QDs had a greater effect on the mitochondrial swelling, membrane fluidity, and permeabilization to H(+) and K(+) by mitochondrial inner membrane, which was caused the fact that BSA was more lipophilic than MPA. This study provides an important basis for understanding the mechanism of the toxicity of CdTe QDs to mitochondria, and valuable information for safe use of QDs in the future.

The interactions between CdSe quantum dots and yeast Saccharomyces cerevisiae: adhesion of quantum dots to the cell surface and the protection effect of ZnS shell.

The interactions between quantum dots (QDs) and biological systems have attracted increasing attention due to concerns on possible toxicity of the nanoscale materials. The biological effects of CdSe QDs and CdSe/ZnS QDs with nearly identical hydrodynamic size on Saccharomyces cerevisiae were investigated via microcalorimetric, spectroscopic and microscopic methods, demonstrating a toxic order CdSe>CdSe/ZnS QDs. CdSe QDs damaged yeast cell wall and reduced the mitochondrial membrane potential. Noteworthy, adhesion of QDs to the yeast cell surface renders this work a good example of interaction site at cell surface, and the epitaxial coating of ZnS could greatly reduce the toxicity of Cd-containing QDs. These results will contribute to the safety evaluation of quantum dots, and provide valuable information for design of nanomaterials.

Mitochondrial dysfunction induced by different concentrations of gadolinium ion.

Gadolinium-based compounds are the most widely used paramagnetic contrast agents in magnetic resonance imaging on the world. But the tricationic gadolinium ion (Gd(3+)) could induce cell apoptosis probably because of its effects on mitochondria. Until now, the mechanism about how Gd(3+) interacts with mitochondria is not well elucidated. In this work, mitochondrial swelling, collapsed transmembrane potential and decreased membrane fluidity were observed to be important factors for mitochondrial permeability transition pore (mtPTP) opening induced by Gd(3+). The protection effect of CsA (Cyclosporin A) could confirm high concentration of Gd(3+) (500 µM) would trigger mtPTP opening. Moreover, mitochondrial outer membrane breakdown and volume expansion observed clearly by transmission electron microscopy and the release of Cyt c (Cytochrome c) could explain the mtPTP opening from another aspect. In addition, MBM(+) (monobromobimane(+)) and DTT (dithiothreitol) could protect thiol (-SH) groups from oxidation so that the toxicity of Gd(3+) might be resulted from the chelation of -SH of membrane proteins by free Gd(3+). Gd(3+) could inhibit the initiation of mitochondrial membrane lipid peroxidation, so it might interact with anionic lipids too. These findings will highly contribute to the safe applications of Gd-based agents.

High concentration of gadolinium ion modifying isolated rice mitochondrial biogenesis.

Mitochondria play an important role in plant growth and development, cooperating with the endoplasmic reticulum and nucleus. Gadolinium, one of the rare earth elements, is an inhibitor of stretch-activated calcium channels located on the endoplasmic reticulum and plasma membrane and has no effect on nuclear calcium variation in plant cells. We analyzed the effects of Gd3+ on mitochondria function by monitoring mitochondrial swelling, changes of membrane fluidity, and transmembrane potential collapse and by observing mitochondrial ultrastructure. We found that high concentration of Gd3+ induces rice mitochondrial dysfunction through mitochondrial permeability transition (MPT). The protection of DTT and EDTA demonstrate that Gd3+ blocks the inner membrane ion channel through thiol chelation.

Ce(III)-induced rice mitochondrial permeability transition investigated by spectroscopic and microscopic studies.

Cerium has been widely used as fertilizer and feed additives in agriculture, but it might finally impair human health by food chain accumulation with its dosage increased in environmental and crops samples. To resolve the conflict, we investigated the effects of Ce(III) on isolated rice mitochondrial permeability transition (MPT) by examining mitochondrial swelling, transmembrane potential, membrane fluidity with spectroscopy, and observing the mitochondrial ultrastructure, meanwhile, the interaction site(s) and mechanism between Ce(III) and mitochondria were also studied. The results showed that the low level of Ce(III) had little effect on rice MPT, however, the higher level of Ce(III) could induce rice MPT, and the thiol (-SH) groups of membrane proteins (defined as "S" site) matched by Ce(III)-triggered rice MPT pore opening.

Microcalorimetric studies of the effect of cerium (Ш) on isolated rice mitochondria fed by pyruvate.

Mitochondria were isolated from the hybrid rice Xiangzaoxian 31, then the effects of low and high concentrations of Ce (Ш) on metabolism of mitochondria fed by pyruvate were investigated respectively, by microcalorimetry and oxygen electrode method The thermogenic curve of mitochondria without Ce (Ш) could be divided into three parts: activity recovery phase, stationary phase and decline phase. And the thermokinetic parameters have been calculated through the metabolic thermogenic curves. With addition of different concentrations of Ce (Ш), the results demonstrated that low levels of cerium ion stimulated the metabolic activity of energized mitochondria and the inhibition was discovered with high concentrations of Ce (Ш). At the same time, it is shown that the effect in respiration correspond to the effect on mitochondrial metabolism on addition of different concentrations of Ce (Ш). Moreover, the addition of low and high concentrations of Ce (Ш) had no obvious effect on the total heat output (Q). The concentration-dependent effect of Ce (Ш) on metabolism of mitochondria is similar to plant growth response to rare earth elements (Hormesis effect).