Two-Photon Time-Gated In Vivo Imaging of Dihydrolipoic-Acid-Decorated Gold Nanoclusters.

Due to the unique advantages of two-photon technology and time-resolved imaging technology in the biomedical field, attention has been paid to them. Gold clusters possess excellent physicochemical properties and low biotoxicity, which make them greatly advantageous in biological imaging, especially for in vivo animal imaging. A gold nanocluster was coupled with dihydrolipoic acid to obtain a functionalized nanoprobe; the material displayed significant features, including a large two-photon absorption cross-section (up to 1.59 × 105 GM) and prolonged fluorescence lifetime (>300 ns). The two-photon and time-resolution techniques were used to perform cell imaging and in vivo imaging.

The Emission Mechanism of Gold Nanoclusters Capped with 11-Mercaptoundecanoic Acid, and the Detection of Methanol in Adulterated Wine Model.

The absorption and emission mechanisms of gold nanoclusters (AuNCs) have yet to be understood. In this article, 11-Mercaptoundecanoic acid (MUA) capped AuNCs (AuNC@MUA) were synthesized using the chemical etching method. Compared with MUA, AuNC@MUA had three obvious absorption peaks at 280 nm, 360 nm, and 390 nm; its photoluminescence excitation (PLE) peak and photoluminescence (PL) peak were located at 285 nm and 600 nm, respectively. The AuNC@MUA was hardly emissive when 360 nm and 390 nm were chosen as excitation wavelengths. The extremely large stokes-shift (>300 nm), and the mismatch between the excitation peaks and absorption peaks of AuNC@MUA, make it a particularly suitable model for studying the emission mechanism. When the ligands were partially removed by a small amount of sodium hypochlorite (NaClO) solution, the absorption peak showed a remarkable rise at 288 nm and declines at 360 nm and 390 nm. These experimental results illustrated that the absorption peak at 288 nm was mainly from metal-to-metal charge transfer (MMCT), while the absorption peaks at 360 nm and 390 nm were mainly from ligand-to-metal charge transfer (LMCT). The PLE peak coincided with the former absorption peak, which implied that the emission of the AuNC@MUA was originally from MMCT. It was also interesting that the emission mechanism could be switched to LMCT from MMCT by decreasing the size of the nanoclusters using 16-mercaptohexadecanoic acid (MHA), which possesses a stronger etching ability. Moreover, due to the different PL intensities of AuNC@MUA in methanol, ethanol, and water, it has been successfully applied in detecting methanol in adulterated wine models (methanol-ethanol-water mixtures).

Super-efficient in Vivo Two-Photon Photodynamic Therapy with a Gold Nanocluster as a Type I Photosensitizer.

Photodynamic therapy (PDT) is a clinically approved, minimally invasive therapeutic technique that can induce the regression of targeted lesions via generating excess cytotoxic reactive oxygen species. However, due to the limited penetration depth of visible excitation light and the intrinsic hypoxia microenvironment of solid tumors, the efficacy of PDT in the treatment of cancer, especially deep-seated or large tumors, is unsatisfactory. Herein, we developed an efficient in vivo PDT system based on a nanomaterial, dihydrolipoic acid coated gold nanocluster (AuNC@DHLA), that combined the advantages of large penetration depth in tissue, extremely high two-photon (TP) absorption cross section (σ2 ∼ 106 GM), efficient ROS generation, a type I photochemical mechanism, and negligible in vivo toxicity. With AuNC@DHLA as the photosensitizer, highly efficient in vivo TP-PDT has been achieved.

A facile synthesis of biocompatible, glycol chitosan shelled CdSeS/ZnS QDs for live cell imaging.

We here report a facile synthesis of chitosan shelled quantum dot (QD/fGC) that holds essential properties requisite for biological applications, such as excellent water solubility, super colloidal stability, and low nonspecific adsorption as well as ease of functionalization. In this method, the amphiphilic glycol chitosan fragment (MW 1.0-1.7 kDa) was assembled on the top of CdSeS/ZnS nanocrystal through hydrophobic interaction in aqueous solution, without displacing the native coordinating ligands, which result in a higher quantum yield of about 0.26, 46% of the uncoated CdSeS/ZnS QDs in chloroform (0.57). In addition, the prepared QD/fGC composes an individual semiconductor core and presents an extremely small size of about 6.03 ± 1.50 nm (n = 399) in diameter. By conjugation with bioactive amines via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-based hydroxyl activation approach, the functionalized QD/fGC presented excellent recognition of specific cells in fluorescent imaging. Our work provides a new general method of chitosan modification of hydrophobic nanoparticles for biomedical applications.

Low impedance nature of 12 acupoints on the limbs, and the unexpected dependence on limb angle.

OBJECTIVE: To better understand the working mechanism of acupuncture, we investigated the skin electrical impedance distribution around acupoints, and the impedance changes at 12 original acupoints bilaterally after bending the limbs. METHODS: We measured the skin electrical impedance in three study subjects in the frequency range of 40 to 10 kHz using the four-electrode method with a sharp probe and a large reference electrode. A measurement matrix of 7 mm ¡Á 7 mm with spacing of 2.0 (or 3.0) mm was measured to obtain 2D impedance mapping of acupoints. The impedance spectra of 12 original acupoints were measured at the 0? position and the 90? position. RESULTS: The electrical impedance of some acupoints, such as Yangchi (TE 4), was 16 times lower than that of the surrounding area, showing a recognizable small central area of low impedance with a diameter of less than 4 mm. In contrast, other acupoints, such as Laogong (PC 8), had an electrical impedance that was not significantly different from that of the surrounding area. When the limb was bent from a straight position (0?) to a vertical position (90?), the electrical impedance of the 12 original acupoints showed varied trends, either increasing or decreasing by a factor of up to ten times, or remaining at the same level. CONCLUSION: Not all acupoints tested show the property of low impedance, which might be related to the varied depth of the openings of superficial collaterals. The unexpected dependence of acupoint impedance on limb angle is a novel discovery, which implies that the channel paths are located in interstitial structures in the limbs. It might be possible to determine an optimized limb position for each particular acupuncture treatment in clinical practice.

Bacteria-mediated in vivo delivery of quantum dots into solid tumor.

Semiconductor nanocrystals, so-called quantum dots (QDs), promise potential application in bioimaging and diagnosis in vitro and in vivo owing to their high-quality photoluminescence and excellent photostability as well as size-tunable spectra. Here, we describe a biocompatible, comparatively safe bacteria-based system that can deliver QDs specifically into solid tumor of living animals. In our strategy, anaerobic bacterium Bifidobacterium bifidum (B. bifidum) that colonizes selectively in hypoxic regions of animal body was successfully used as a vehicle to load with QDs and transported into the deep tissue of solid tumors. The internalization of lipid-encapsuled QDs into B. bifidum was conveniently carried by electroporation. To improve the efficacy and specificity of tumor targeting, the QDs-carrying bacterium surface was further conjugated with folic acids (FAs) that can bind to the folic acid receptor overexpressed tumor cells. This new approach opens a pathway for delivering different types of functional cargos such as nanoparticles and drugs into solid tumor of live animals for imaging, diagnosis and therapy.

The role of calcium ions in the interactions of PrP106-126 amide with model membranes.

In this work, we investigated the interactions of PrP106-126 amide with 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) and POPC/bovine brain sphingomyelin (BSM) membranes in the presence of calcium ions by in situ time-lapse atomic force microscopy (AFM) and circular dichroism (CD). The CD results show that Ca(2+) has no obvious effects on the random coil conformation of PrP106-126 amide. The tapping mode AFM results demonstrate that electrostatic interaction decreases the measured heights of supported lipid bilayers (SLBs) in HBS-Ca(2+) solution. Electrostatic interaction analysis also can be used to determine the applied force in liquid tapping mode AFM. The interactions of PrP106-126 amide with membranes by AFM demonstrate the following: (i) Ca(2+) inhibits the interaction of PrP106-126 amide with POPC lipid and (ii) the co-interaction between Ca(2+) and BSM increases the poration ability of PrP106-126 amide. These results imply that the main role of Ca(2+) in the interactions of PrP106-126 amide with membranes is changing the surface properties of the membranes.

Polyvalent lactose-quantum dot conjugate for fluorescent labeling of live leukocytes.

Oligosaccharides play crucial roles in many biorecognition processes by the so-called "cluster glycosidic effect". We here report a facile synthesis of lactose-CdSeS/ZnS quantum dot conjugate (Lac-QDs) by use of 1-thiol-beta-D-lactose via ligand exchange, which exhibits significantly high affinity and specificity to leukocytes in contrast to the monovalent lactose. Structural analyses indicate that there are about 132 lactosyl molecules assembled on single QDs and the hydrodynamic diameter is small, close to 8.2 nm. Further, Lac-QDs display good fluorescence and physicochemical stability in physiological conditions, as well as extremely low cytotoxicity. These properties facilitate the use of Lac-QDs in fluorescent labeling of live leukocytes.

A facile synthesis of small-sized, highly photoluminescent, and monodisperse CdSeS QD/SiO(2) for live cell imaging.

In recent years, silica coating has been extensively investigated to fabricate the biocompatible interface of quantum dots (QDs) for biomedical applications. We here describe a facile and efficient method of synthesizing high-quality silica-coated CdSeS QDs (CdSeS QD/SiO(2)), where an immediate photoluminescence-favorable microenvironment is first created by assembling amphiphilic molecules around the CdSeS core, and a thin silica shell is further introduced to protect this hydrophobic interlayer. The prepared CdSeS QD/SiO(2) exhibits excellent properties such as good water solubility, low cytotoxicity, and high quantum yield (QY, up to 0.49) as well as the resistance of photobleaching in aqueous solution. Also, the CdSeS QD/SiO(2) nanoparticles homogeneously comprise single CdSeS cores and hold a comparatively small size up to about 11 nm in diameter. Particularly, this method leads to a significant increase in QY as compared to the uncoated CdSeS QDs ( approximately 109% of the initial QY), though only thin silica shells formed in the CdSeS QD/SiO(2) structure. By coupling with folic acids, the CdSeS QD/SiO(2) conjugates were successfully used for tumor cell labeling. Our results demonstrated a robust hydrophobic QDs-based approach for preparing highly photoluminescent, biocompatible QD/SiO(2) through creation of a stable hydrophobic interlayer surrounding the QD cores, which could be also suitable for silica coating of other kinds of hydrophobic nanoparticles.

Effects of lipid composition and phase on the membrane interaction of the prion peptide 106-126 amide.

Lipid rafts are specialized liquid-ordered (L(o)) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (L(d)) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an L(d)-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an L(d)-L(o) phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K(106)TNMKHMAGAAAAGAVVGGLG(126) (PrP106-126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the L(d) phase, the PrP106-126 amide induces fully penetrated porations in the L(d) phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the L(d) phase, the PrP106-126 amide induces the disintegration of the L(d) phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the L(o) phase, PrP106-126 amide induces membrane thinning in the L(o) phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106-126 amide interacts with lipid membranes.

PrP106-126 peptide disrupts lipid membranes: influence of C-terminal amidation.

PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP(C) to scrapie isoform PrP(Sc)). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126(CONH2) and PrP106-126(COOH)) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126(COOH) displayed a higher peptide-lipid binding affinity than PrP106-126(CONH2) (approximately 2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.

Parallel-oriented fibrogenesis of a beta-sheet forming peptide on supported lipid bilayers.

Peptide self-assembly on substrates is currently an intensively studied topic that provides a promising strategy for fabrication of soft materials and is also important for revealing the surface chemistry of amyloidogenic proteins that aggregate on cell membranes. We investigated the fibrogenesis of a beta-sheet forming peptide Abeta(26-35) on supported lipid bilayers (SLBs) by in situ atomic force microscopy (AFM), circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results show that the Abeta(26-35) nanofilaments' growth is oriented to a specific direction and formed a highly ordered, large-scale, parallel-oriented surface pattern on membranes. The parallel-oriented fibrogenesis of Abeta(26-35) was able to occur on different lipid membranes rather than on solid substrates. It implies that the parallel-oriented fibrogenesis was associated with the distinct properties of lipid membranes, such as the fluid nature of lipid molecules on membranes. The membrane fluidity may allow the peptide assemblies to float at the water-membrane interface and easily orient to an energetically favorable state. These results provide an insight into the surface chemistry of peptide self-assembly on lipid membranes and highlight a possible way to fabricate supramolecular architectures on the surface of soft materials.

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers.

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP(Sc), an abnormally misfolded, protease-resistant, and beta-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP(Sc) and presents similar neurotoxicity as PrP(Sc). By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.

One-dimensional self-assembly of a rational designed beta-structure peptide.

Fabricating various nanostructures based on the self-assembly of diverse biological molecules is now of great interest to the field of bionanotechnology. In this study, we report a de novo designed peptide (T1) with a preferential beta-hairpin forming property that can spontaneously assemble into nanofibrils in ultrapure water. The nanofibrils assembled by T1 could grow up to tens of microns in length with a left-handed helical twist and an average height of 4.9 +/- 0.9 nm. Moreover, protofilaments and nucleus structures both with a similar height of 1.4 +/- 0.2 nm were observed during fibrilization as well as via sonication of the mature nanofibrils. A typical conformational transition from random coil to beta-structure was observed in association with the fibrilization. Molecular modeling of T1 assemblies displayed that the beta-hairpin molecules organize in a parallel fashion in which the beta-strands align in an antiparallel fashion and each adjoining beta-strand runs left-handed twist at about 2.9 degrees with respect to the one located before it along the fibrillar axis. It also revealed that the maximum thickness of the assembly intermediate, the helical tape structure, is about 1.4 nm and four tapes can further assemble into a fibril with a diameter of about 4.1 nm. Taken together the results obtained by AFM, CD, and molecular modeling, T1 fibrilization probably undergoes a hierarchy approach, in which the aromatic stacking and the electrostatic interactions between the assembled structures are most likely the two major factors directing the one-dimensional self-assembly. Based on these studies, we propose T1 can be used as a model peptide to investigate the beta-sheet based self-assembly process and could be a potential bioorganic template to develop functional materials.

Osmophobic effect of glycerol on irreversible thermal denaturation of rabbit creatine kinase.

Protein stability plays an extremely important role not only in its biological function but also in medical science and protein engineering. Osmolytes provide a general method to protect proteins from the unfolding and aggregation induced by extreme environmental stress. In this study, the effect of glycerol on protection of the model enzyme creatine kinase (CK) against heat stress was investigated by a combination of spectroscopic method and thermodynamic analysis. Glycerol could prevent CK from thermal inactivation and aggregation in a concentration-dependent manner. The spectroscopic measurements suggested that the protective effect of glycerol was a result of enhancing the structural stability of native CK. A further thermodynamic analysis using the activated-complex theory suggested that the effect of glycerol on preventing CK against aggregation was consistent with those previously established mechanisms in reversible systems. The osmophobic effect of glycerol, which preferentially raised the free energy of the activated complex, shifted the equilibrium between the native state and the activated complex in favor of the native state. A comparison of the inactivation rate and the denaturation rate suggested that the protection of enzyme activity by glycerol should be attributed to the enhancement of the structural stability of the whole protein rather than the flexible active site.