RESUMO
Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross-α amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic resolution, an architecture initially discovered in the bacterial PSMα3 cytotoxin. The fibrils of uperin 3.5 and PSMα3 comprised antiparallel and parallel helical sheets, respectively, recapitulating properties of ß-sheets. Uperin 3.5 demonstrated chameleon properties of a secondary structure switch, forming mostly cross-ß fibrils in the absence of lipids. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-α/ß amyloid fibrils.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Citotoxinas/química , Citotoxinas/metabolismo , Cinética , Lagartos/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus hominis/efeitos dos fármacos , Homologia Estrutural de ProteínaRESUMO
Multispectral photodetectors (MSPs) and circularly polarized light (CPL) sensors are important in opto-electronics, photonics, and imaging. A capacitive photodetector consisting of an interdigitated electrode coated with carbon dot/anthraquinone-polydiacetylene is constructed. Photoexcitation of the carbon dots induces transient electron transfer to the anthraquinone moieties, and concomitant change in the film dielectric constant and recorded capacitance. This unique photodetection mechanism furnishes wavelength selectivity that is solely determined by the absorbance of the carbon dots incorporated in the anthraquinone-polydiacetylene matrix. Accordingly, employing an array of polymerized-anthraquinone photodetector films comprising carbon dots (C-dots) exhibiting different excitation wavelengths yielded optical "capacitive fingerprints" in a broad spectral range (350-650 nm). Furthermore, circular light polarization selectivity is achieved through chiral polymerization of the polydiacetylene framework. The carbon dot/anthraquinone-polydiacetylene capacitive photodetector features rapid photo-response, high fidelity, and recyclability as the redox reactions of anthraquinone are fully reversible. The carbon dot/anthraquinone-polydiacetylene platform is inexpensive, easy to fabricate, and consists of environmentally friendly materials.
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High-resolution structures are crucial for understanding the functional properties of nanomaterials. We applied single-particle cryo-electron microscopy (cryo-EM), a method traditionally used for structure determination of biological macromolecules, to obtain high-resolution structures of synthetic non-biological filaments formed by photopolymerization of macrocyclic diacetylene (MDA) amphiphilic monomers. Tomographic analysis showed that the MDA monomers self-assemble into hollow nanotubes upon dispersion in water. Single-particle analysis revealed tubes consisting of six pairs of covalently bonded filaments held together by hydrophobic interactions, where each filament is composed of macrocyclic rings stacked in parallel "chair" conformations. The hollow MDA nanotube structures we found may account for the efficient scavenging of amphiphilic pollutants in water and subsequent photodegradation of the guest species.
Assuntos
Poluentes Ambientais , Nanotubos , Microscopia Crioeletrônica/métodos , Polímero Poliacetilênico , ÁguaRESUMO
Resveratrol, a natural polyphenol, exhibits beneficial health properties and has been touted as a potential anti-tumor agent. Here, we demonstrate potent anti-cancer effects of carbon dots (C-dots) synthesized from resveratrol. The mild synthesis conditions retained resveratrol functional moieties upon the carbon dots' (C-dots) surface, an important requisite for achieving specificity toward cancer cells and biological activities. Indeed, the disruptive effects of the resveratrol-C-dot were more pronounced in several cancer cell types compared to normal cells, underscoring targeting capabilities of the C-dots, a pertinent issue for the development of cancer therapeutics. In particular, we observed impairment of mitochondrial functionalities, including intracellular calcium release, inhibition of cytochrome-C oxidase enzyme activity, and mitochondrial membrane perturbation. Furthermore, the resveratrol C-dots were more potent than either resveratrol molecules alone, known anti-cancer polyphenolic agents such as curcumin and triphenylphosphonium, or C-dots prepared from different carbonaceous precursors. This study suggests that resveratrol-synthesized C-dots may have promising therapeutic potential as anti-cancer agents.
Assuntos
Antineoplásicos , Curcumina , Neoplasias , Estilbenos , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose , Cálcio/metabolismo , Carbono/metabolismo , Curcumina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/farmacologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Polifenóis/metabolismo , Polifenóis/farmacologia , Resveratrol/farmacologia , Estilbenos/farmacologiaRESUMO
BIM is a key apoptotic protein, participating in diverse cellular processes. Interestingly, recent studies have hypothesized that BIM is associated with the extensive neuronal cell death encountered in protein misfolding diseases, such as Alzheimer's disease. Here, we report that the core pro-apoptotic domain of BIM, the BIM-BH3 motif, forms ubiquitous amyloid fibrils. The BIM-BH3 fibrils exhibit cytotoxicity, disrupt mitochondrial functions, and modulate the structures and dynamics of mitochondrial membrane mimics. Interestingly, a slightly longer peptide in which BIM-BH3 was flanked by four additional residues, widely employed as a model of the pro-apoptotic core domain of BIM, did not form fibrils, nor exhibited cell disruptive properties. The experimental data suggest a new mechanistic role for the BIM-BH3 domain, and demonstrate, for the first time, that an apoptotic peptide forms toxic amyloid fibrils.
Assuntos
Amiloide/química , Apoptose , Proteína 11 Semelhante a Bcl-2/química , Domínios Proteicos , Sequência de Aminoácidos , Amiloide/metabolismo , Amiloide/ultraestrutura , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dicroísmo Circular , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Creation of new two-dimensional (2D) architectures has attracted significant attention in the field of self-assembly for structural diversity and new functionalization. Although numerous 2D polymer nanosheets have been reported, 2D nanosheets with tubular channels have been unexplored. Herein, we describe a new strategy for the fabrication of stimulus-responsive conjugated polymer 2D nanosheets with hollow cavities. Amphiphilic macrocyclic diacetylenes self-assembled in an aqueous solution in a columnar manner to afford bilayered 2D nanosheets with intrinsically tubular nanochannels. UV-induced polymerization resulted in the generation of blue-colored tubular conjugated polydiacetylene 2D nanosheets. Immobilization of gold nanoparticles, fluorescence labeling with FRET phenomenon and colorimetric DNA sensing were demonstrated with these new 2D nanosheets. In addition, the free NH2 containing polymerized 2D nanosheet was utilized for conductivity behavior and grafting on graphene oxide (GO).
Assuntos
Nanopartículas Metálicas , Polímeros Responsivos a Estímulos , Ouro , Polímeros/química , ColorimetriaRESUMO
Neurofibrillary tangles of the Tau protein and plaques of the amyloid ß peptide are hallmarks of Alzheimer's disease (AD), which is characterized by the conversion of monomeric proteins/peptides into misfolded ß-sheet rich fibrils. Halting the fibrillation process and disrupting the existing aggregates are key challenges for AD drug development. Previously, we performed in vitro high-throughput screening for the identification of potent inhibitors of Tau aggregation using a proxy model, a highly aggregation-prone hexapeptide fragment 306VQIVYK311 (termed PHF6) derived from Tau. Here we have characterized a hit molecule from that screen as a modulator of Tau aggregation using in vitro, in silico, and in vivo techniques. This molecule, an anthraquinone derivative named Purpurin, inhibited ~ 50% of PHF6 fibrillization in vitro at equimolar concentration and disassembled pre-formed PHF6 fibrils. In silico studies showed that Purpurin interacted with key residues of PHF6, which are responsible for maintaining its ß-sheets conformation. Isothermal titration calorimetry and surface plasmon resonance experiments with PHF6 and full-length Tau (FL-Tau), respectively, indicated that Purpurin interacted with PHF6 predominantly via hydrophobic contacts and displayed a dose-dependent complexation with FL-Tau. Purpurin was non-toxic when fed to Drosophila and it significantly ameliorated the AD-related neurotoxic symptoms of transgenic flies expressing WT-FL human Tau (hTau) plausibly by inhibiting Tau accumulation and reducing Tau phosphorylation. Purpurin also reduced hTau accumulation in cell culture overexpressing hTau. Importantly, Purpurin efficiently crossed an in vitro human blood-brain barrier model. Our findings suggest that Purpurin could be a potential lead molecule for AD therapeutics.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antraquinonas/farmacologia , Oligopeptídeos/genética , Agregados Proteicos/efeitos dos fármacos , Proteínas tau/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Animais Geneticamente Modificados/genética , Barreira Hematoencefálica/efeitos dos fármacos , Modelos Animais de Doenças , Drosophila melanogaster/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Conformação Proteica em Folha beta/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genéticaRESUMO
Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as in amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time, to our knowledge, imaging flow cytometry for investigating interactions of representative amyloidogenic peptides, namely, the 106-126 fragment of prion protein (PrP(106-126)) and the human islet amyloid polypeptide (hIAPP), with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106-126)-membrane interactions by epigallocatechin gallate, a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106-126) and hIAPP. Specifically, the experiments reveal that both PrP(106-126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that epigallocatechin gallate ameliorated vesicle disruption by PrP(106-126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas , Príons , Amiloide , Proteínas Amiloidogênicas , Citometria de Fluxo , HumanosRESUMO
Rational engineering of highly stable and Raman-active nanostructured substrates is still urgently in demand for achieving sensitive and reliable surface-enhanced Raman spectroscopy (SERS) analysis in solution phase. Herein, monodisperse N-doping graphene quantum dots wrapped Au nanoparticles (Au-NGQD NPs) were facilely prepared, and further their applications as substrates in SERS-based detection and cellular imaging have been explored. The as-prepared Au-NGQD NPs exhibit superior long-term stability and biocompatibility, as well as large enhancement capability due to the integration of electromagnetic and chemical enhancements. The practical applicability of the Au-NGQD NPs was verified via the direct SERS tests of several kinds of aromatics in solution phase. Finite-difference time-domain simulations in combination with density functional theory calculation were also successfully used to explain the enhancement mechanism. Furthermore, the Au-NGQD NPs were conjugated with 4-nitrobenzenethiol (4-NBT, as reporter) and 4-mercaptophenylboronic acid (MPBA, as targeting element) to construct the MPBA/4-NBT@Au-NGQD probes, which could specifically recognize glycan-overexpressed cancer cells through SERS imaging on a cell surface. The prepared Au-NGQDs show great potential as superior SERS substrates in solution phase for on-site Raman detection.
RESUMO
Because of their unique structural and optical properties, 1D perylene diimide (PDI) derivatives have gained attention for use in optoelectronic devices. However, PDI-containing self-assembled supramolecular systems often are of limited use because they have supramolecular architectures that are held together by weak noncovalent π-π stacking, hydrogen bonding, and hydrophobic interactions. As a result, they are intrinsically unstable under solution-processing conditions. To overcome this limitation, a polydiacetylene (PDA)-based strategy is developed to construct a solvent-resistant and stable PDI assembly. For this purpose, first the monomer PDI-BisDA is generated, in which two polymerizable diacetylene (DA) units are covalently linked to a PDI core. Importantly, 254 nm UV irradiation of self-assembled PDI-BisDA nanofibers forms solvent-resistant and stable PDI-PDA fibers. Owing to the presence of PDA, the generated polymer fibers display an increased photocurrent. In addition, the existence of PDA and PDI moieties in the fiber leads to the occurrence of switchable on-off fluorescence resonance energy transfer (FRET) between the PDI and reversibly thermochromic PDA chromophores.
RESUMO
Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different ß sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.
Assuntos
Amiloide/química , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biofilmes , Membrana Celular/química , Membranas Artificiais , Substituição de Aminoácidos , Amiloide/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana , Multimerização ProteicaRESUMO
2D carbon and nitrogen based semiconductors (CN) have attracted widespread attention for their possible use as low-cost and environmentally friendly materials for various applications. However, their limited solution-dispersibility and the difficulty in preparing exfoliated sheets with tunable photophysical properties restrain their exploitation in imaging-related applications. Here, the synthesis of carbon and nitrogen organic scaffolds with highly tunable optical properties, excellent dispersion in water and DMSO, and good bioimaging properties is reported. Tailored photophysical and chemical properties are acquired by the synthesis of new starting monomers containing different substituent chemical groups with varying electronic properties. Upon monomer condensation at moderate temperature, 350 °C, the starting chemical groups are fully preserved in the final CN. The low condensation temperature and the effective molecular-level modification of the CN scaffold lead to well-dispersed photoluminescent CN thin sheets with a wide range of emission wavelengths. The good bioimaging properties and the tunable fluorescence properties are exemplified by in situ visualization of giant unilamellar vesicles in a buffered aqueous solution as a model system. This approach opens the possibility for the design of tailor-made CN materials with tunable photophysical and chemical properties toward their exploitation in various fields, such as photocatalysis, bioimaging, and sensing.
RESUMO
Assembly of fibrillar peptide structures is dependent both upon their intrinsic propensities toward ß-structure formation, as well as on structural modulation by external molecular factors. ß-sheet structures may either be designed to form useful assemblies or be the undesired consequence of protein denaturation to toxic amyloid structures in several neurodegenerative diseases. Membrane bilayers have been implicated as primary initiators and modulators of amyloid fibrillation and the reasons for this effect are yet to be elucidated. Here, we employed a set of three charged peptides having the tendency to form ß-sheet fibrils, to investigate the effect of zwitterionic and negatively charged bilayer vesicles on their assembly structures. Microscopic and spectroscopic experiments revealed intimate relationship between peptide/membrane charges and fibrillation properties. Electrostatic attraction was apparent between oppositely charged peptides and vesicles; however, such interactions did not appear to significantly modulate fibril morphologies of either the net anionic peptide or the cationic one. Yet, a dramatic structural effect was observed when the nominal zwitterionic peptide underwent fibrillation in the presence of negatively charged vesicles. Assemblies of this peptide display a net positive charge, which facilitated the counterionic interactions with the vesicles. Furthermore, these interactions templated a unique twisted fiber morphology demonstrating the dramatic effect membrane-mediated interactions exert on fibril morphologies.
Assuntos
Amiloide/química , Bicamadas Lipídicas/química , Conformação Proteica em Folha beta , Eletricidade EstáticaRESUMO
PAP248â»286, a 39 amino acid peptide fragment, derived from the prostatic acid phosphatase secreted in human semen, forms amyloid fibrils and facilitates the attachment of retroviruses to host cells that results in the enhancement of viral infection. Therefore, the inhibition of amyloid formation by PAP248â»286 (termed PAP f39) may likely reduce HIV transmission in AIDS. In this study, we show that the naphthoquinone tryptophan (NQTrp) hybrid molecule significantly inhibited PAP f39 aggregation in vitro in a dose-dependent manner as observed from the ThT assay, ANS assay, and transmission electron microscopy imaging. We found that even at a sub-molar concentration of 20:1 [PAP f39:NQTrp], NQTrp could reduce >50% amyloid formation. NQTrp inhibition of PAP f39 aggregation resulted in non-toxic intermediate species as determined by the vesicle leakage assay. Isothermal titration calorimetry and molecular docking revealed that the binding of NQTrp and PAP f39 is spontaneous, and NQTrp predominantly interacts with the polar and charged residues of the peptide by forming hydrogen bonds and hydrophobic contacts with a strong binding energy. Collectively, these findings indicate that NQTrp holds significant potential as a small molecule inhibitor of semen amyloids.
Assuntos
Fosfatase Ácida/metabolismo , Amiloide/metabolismo , Naftoquinonas/química , Naftoquinonas/farmacologia , Fosfatase Ácida/química , Amiloide/antagonistas & inibidores , Sítios de Ligação , Corantes/química , Vermelho Congo/química , Relação Dose-Resposta a Droga , Simulação de Acoplamento Molecular , Naftoquinonas/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Termodinâmica , Triptofano/químicaRESUMO
Bacosides, class of compounds extracted from the Bacopa monniera plant, exhibit interesting therapeutic properties, particularly enhancing cognitive functions and putative anti-amyloid activity. We show that bacoside-A exerted significant effects upon fibrillation and membrane interactions of the amyloidogenic fragment of the prion protein [PrP(106-126)]. Specifically, when co-incubated with PrP(106-126), bacoside-A accelerated fibril formation in the presence of lipid bilayers and in parallel inhibited bilayer interactions of the peptide aggregates formed in solution. These interesting phenomena were studied by spectroscopic and microscopic techniques, which suggest that bacoside A-promoted fibrillation reduced the concentration of membrane-active pre-fibrillar species of the prion fragment. This study suggests that induction of fibril formation and corresponding inhibition of membrane interactions are likely the underlying factors for ameliorating amyloid protein toxicity by bacoside-A.
Assuntos
Amiloide/química , Membranas Artificiais , Proteínas Priônicas/química , Saponinas/química , Triterpenos/química , Amiloide/antagonistas & inibidores , Proteínas Priônicas/antagonistas & inibidoresRESUMO
Detection of reactive oxygen species (ROS) is important in varied biological processes, disease diagnostics, and chemotherapeutic drug screening. We constructed a ROS sensor comprising an ascorbic-acid-based hydrogel encapsulating luminescent amphiphilic carbon-dots (C-dots). The sensing mechanism is based upon ROS-induced oxidation of the ascorbic acid units within the hydrogel scaffold; as a consequence, the hydrogel framework collapses resulting in aggregation of the C-dots and quenching of their luminescence. The C-dot-hydrogel platform exhibits high sensitivity and detected ROS generated chemically in solution and in actual cell environments. We demonstrate application of the C-dot-hydrogel for evaluating the efficacy of a chemotherapeutic substance, underscoring the potential of the system for drug screening applications.
Assuntos
Ácido Ascórbico/química , Carbono/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Espécies Reativas de Oxigênio/análise , Morte Celular , Citometria de Fluxo , Células HeLa , Humanos , Conformação Molecular , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
Multifunctional nanoparticles have attracted significant interest as biomedical vehicles, combining diagnostic, imaging, and therapeutic properties. We describe herein the construction of new nanoparticle conjugates comprising WS2 nanorods (NRs) coupled to fluorescent carbon dots (C-dots). We show that the WS2 -C-dot hybrids integrate the unique physical properties of the two species, specifically the photothermal activity of the WS2 NRs upon irradiation with near-infrared (NIR) light and the excitation-dependent luminescence emission of the C-dots. The WS2 -C-dot NRs have been shown to be non-cytotoxic and have been successfully employed for multicolour cell imaging and targeted cell killing under NIR irradiation, pointing to their potential utilization as effective therapeutic vehicles.
Assuntos
Carbono/química , Nanotubos/química , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Corantes Fluorescentes/química , Células HeLa , Humanos , Raios Infravermelhos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanotubos/toxicidade , Espectroscopia Fotoeletrônica , Fototerapia , Análise Espectral Raman , Nanomedicina Teranóstica , Tungstênio/químicaRESUMO
Elucidating the dynamic properties of membranes is important for understanding fundamental cellular processes and for shedding light on the interactions of proteins, drugs, and viruses with the cell surface. Dynamic studies of lipid bilayers have been constrained, however, by the relatively small number of pertinent molecular probes and the limited physicochemical properties of the probes. We show that a lipid conjugate comprised of a fluorescent carbon dot (C-dot) covalently attached to a phospholipid constitutes a versatile and effective vehicle for studying bilayer dynamics. The C-dot-modified phospholipids readily incorporated within biomimetic membranes, including solid-supported bilayers and small and giant vesicles, and inserted into actual cellular membranes. We employed the C-dot-phospholipid probe to elucidate the effects of polymyxin-B (a cytolytic peptide), valproic acid (a lipophilic drug), and amyloid-ß (a peptide associated with Alzheimer's disease) upon bilayer fluidity and lipid dynamics through the application of various biophysical techniques.
Assuntos
Carbono/química , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Nanopartículas/química , Fosfolipídeos/química , Membrana Celular/química , Membrana Celular/metabolismo , Espectrometria de FluorescênciaRESUMO
Development of new imaging tools for cancer cells in vitro and in vitro is important for advancing cancer research, elucidating drug effects upon cancer cells, and studying cellular processes. We showed that fluorescent carbon dots (C-dots) synthesized from folic acid can serve as an effective vehicle for imaging cancer cells expressing the folate receptor on their surface. The C-dots, synthesized through a simple one-step process from folic acid as the carbon source, exhibited selectivity towards cancer cells displaying the folate receptor, making such cells easily distinguishable in fluorescence microscopy imaging. Biophysical measurements and competition experiments both confirmed the specific targeting and enhanced uptake of C-dots by the folate receptor-expressing cells. The folic acid-derived C-dots were not cytotoxic, and their use in bioimaging applications could aid biological studies of cancer cells, identification of agonists/antagonists, and cancer diagnostics.
Assuntos
Carbono/química , Receptor 1 de Folato/metabolismo , Imagem Óptica , Pontos Quânticos/química , Sobrevivência Celular , Receptor 1 de Folato/química , Ácido Fólico/química , Células HeLa , Humanos , Microscopia Confocal , Microscopia de FluorescênciaRESUMO
At high concentrations, polyphenols induce cell death, and the polyphenols-rich pomegranate juice (PJ), known for its antioxidative/antiatherogenic properties, can possibly affect cell death, including macrophage death involved in atherogenesis. In the present study, apoptotic/necrotic macrophage death was analyzed in J774A.1 macrophages and in peritoneal macrophages isolated from atherosclerotic apoE-/- mice treated with PJ. The effects of PJ were compared with those of the free radical generator 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Both PJ and AAPH significantly increased J774A.1 macrophage death; however, flow cytometric and microscopic analyses using annexin V/propidium iodide revealed that PJ increased the early apoptosis of the macrophage dose dependently (up to 2.5-fold, P < 0.01), whereas AAPH caused dose-dependent increases in late apoptosis/necrosis (up to 12-fold, P < 0.001). Unlike PJ, AAPH-induced macrophage death was associated with increased intracellular oxidative stress (up to 7-fold, P < 0.001) and with lipid stress demonstrated by triglyceride accumulation (up to 3-fold, P < 0.01) and greater chromatic vesicle response to culture medium (up to 5-fold, P < 0.001). Accordingly, recombinant paraoxonase 1, which hydrolyzes oxidized lipids, attenuated macrophage death induced by AAPH, but not by PJ. Similar apoptotic and oxidative effects were found in macrophages from apoE-/- mice treated with PJ or AAPH. As macrophage apoptotic/necrotic death has considerable impact on atherosclerosis progression, these findings may provide novel mechanisms for the antiatherogenicity of PJ.