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1.
Int J Nanomedicine ; 15: 3071-3085, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431502

RESUMO

Purpose: Recently, two-dimensional (2D) nanomaterials are gaining tremendous attention as novel antibacterial platforms to combat against continuously evolving antimicrobial resistance levels. Among the family of 2D nanomaterials, black phosphorus (BP) nanosheets have demonstrated promising potential for biomedical applications. However, there is a need to gain nanoscale insights of the antibacterial activity of BP nanosheets which lies at the center of technical challenges. Methods: Ultra-large BP nanosheets were synthesized by liquid-exfoliation method in the eco-friendly deoxygenated water. Synthesized BP nanosheets were characterized by TEM, AFM, and Raman spectroscopy techniques and their chemical stability was evaluated by EDS and EELS elemental analysis. The antibacterial activity of BP nanosheets was evaluated at nanoscale by the ultramicrotome TEM technique. Further, HAADF-STEM image and EDS elemental line map of the damaged bacterium were utilized to analyze the presence of diagnostic ions. Supportive SEM and ATR-FTIR studies were carried out to confirm the bacterial cell wall damage. In vitro colony counting method was utilized to evaluate the antibacterial performance of ultra-large BP nanosheets. Results: Elemental EELS and EDS analysis of BP nanosheets stored in deoxygenated water confirmed the absence of oxygen peak. TEM studies indicate the various events of bacterial cell damage with the lost cellular metabolism and structural integrity. Colony counting test results show that as-synthesized BP nanosheets (100 µg/mL) can kill ~95% bacteria within 12 hours. Conclusion: TEM studies demonstrate the various events of E. coli membrane damage and the loss of structural integrity. These events include the BP nanosheets interaction with the bacterial cell wall, cytoplasmic leakage, detachment of cytoplasm from the cell membrane, reduced density of lipid bilayer and agglomerated DNA structure. The EDS elemental line mapping of the damaged bacterium confirms the disrupted cell membrane permeability and the lost cellular metabolism. SEM micrographs and ATR-FTIR supportive results confirm the bacterial cell wall damage.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Nanoestruturas/química , Fósforo/química , Parede Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Escherichia coli/ultraestrutura , Testes de Sensibilidade Microbiana , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Água/química
2.
Int J Nanomedicine ; 15: 1929-1938, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32256069

RESUMO

Background: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms. Methods and Results: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope. Conclusion: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Portadores de Fármacos/química , Membrana Externa Bacteriana/ultraestrutura , Portadores de Fármacos/farmacologia , Escherichia coli/efeitos dos fármacos , Microplásticos/química , Microplásticos/farmacologia , Microscopia Eletrônica de Transmissão , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Propriedades de Superfície
3.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 2): 58-64, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32039886

RESUMO

Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to the well characterized AAVrh.10 serotype, for which no structure is available, is greater than 98%. Here, the structure of the AAVhu.37 capsid at 2.56 Šresolution obtained via single-particle cryo-electron microscopy is presented.

4.
Nanoscale ; 11(36): 16868-16878, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31482911

RESUMO

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.


Assuntos
Compostos Férricos/metabolismo , Microscopia Eletrônica de Transmissão e Varredura , Miocárdio , Baço , Ferritinas , Grafite , Humanos , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Baço/metabolismo , Baço/ultraestrutura
5.
Int J Nanomedicine ; 14: 371-382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30662261

RESUMO

Background: Islet cell transplantation is one of the key treatments for type 1 diabetes. Understanding the mechanisms of insulin fusion and exocytosis are of utmost importance for the improvement of the current islet cell transplantation and treatment of diabetes. These phenomena have not been fully evaluated due either to the lack of proper dynamic imaging, or the lack of proper cell preservation during imaging at nanoscales. Methods: By maintaining the native environment of pancreatic ß-cells between two graphene monolayer sheets, we were able to monitor the subcellular events using in situ graphene liquid cell (GLC)-transmission electron microscopy (TEM) with both high temporal and high spatial resolution. Results: For the first time, the nucleation and growth of insulin particles until the later stages of fusion were imaged at nanometer scales. The release of insulin from plasma membrane involves the degradation of plasma membrane and drastic reductions in the shorter axis of the insulin particles. Sequential exocytosis results indicated the nucleation, growth and attachment of the new insulin particles to the already anchored ones, which is thermodynamically favorable due to the reduction in total surface, further reducing the Gibbs free energy. The retraction of the already anchored insulin toward the cell is also monitored for the first time live at nanoscale resolution. Conclusion: Investigation of insulin granule dynamics in ß-cells can be investigated via GLC-TEM. Our findings with this technology open new realms for the development of novel drugs on pathological pancreatic ß-cells, because this approach facilitates observing the effects of the stimuli on the live cells and insulin granules.


Assuntos
Grafite/química , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Microscopia Eletrônica de Transmissão , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Exocitose , Insulina/metabolismo , Fusão de Membrana , Camundongos
6.
Nanoscale ; 11(2): 698-705, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30565643

RESUMO

Understanding the biomineralization pathways in living biological species is a grand challenge owing to the difficulties in monitoring the mineralization process at sub-nanometer scales. Here, we monitored the nucleation and growth of magnetosome nanoparticles in bacteria and in real time using a transmission electron microscope (TEM). To enable biomineralization within the bacteria, we subcultured magnetotactic bacteria grown in iron-depleted medium and then mixed them with iron-rich medium within graphene liquid cells (GLCs) right before imaging the bacteria under the microscope. Using in situ electron energy loss spectroscopy (EELS), the oxidation state of iron in the biomineralized magnetosome was analysed to be magnetite with trace amount of hematite. The increase of mass density of biomineralized magnetosomes as a function of incubation time indicated that the bacteria maintained their functionality during the in situ TEM imaging. Our results underpin that GLCs enables a new platform to observe biomineralization events in living biological species at unprecedented spatial resolution. Understanding the biomineralization processes in living organisms facilitates the design of biomimetic materials, and will enable a paradigm shift in understanding the evolution of biological species.


Assuntos
Biomineralização/fisiologia , Grafite/química , Magnetossomos/química , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Meios de Cultura/química , Compostos Férricos/metabolismo , Óxido Ferroso-Férrico/metabolismo , Ferro/química , Ferro/metabolismo , Magnetossomos/ultraestrutura , Magnetospirillum/ultraestrutura , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Espectroscopia de Perda de Energia de Elétrons
7.
Nat Commun ; 9(1): 624, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29434200

RESUMO

Nanocarrier administration has primarily been restricted to intermittent bolus injections with limited available options for sustained delivery in vivo. Here, we demonstrate that cylinder-to-sphere transitions of self-assembled filomicelle (FM) scaffolds can be employed for sustained delivery of monodisperse micellar nanocarriers with improved bioresorptive capacity and modularity for customization. Modular assembly of FMs from diverse block copolymer (BCP) chemistries allows in situ gelation into hydrogel scaffolds following subcutaneous injection into mice. Upon photo-oxidation or physiological oxidation, molecular payloads within FMs transfer to micellar vehicles during the morphological transition, as verified in vitro by electron microscopy and in vivo by flow cytometry. FMs composed of multiple distinct BCP fluorescent conjugates permit multimodal analysis of the scaffold's non-inflammatory bioresorption and micellar delivery to immune cell populations for one month. These scaffolds exhibit highly efficient bioresorption wherein all components participate in retention and transport of therapeutics, presenting previously unexplored mechanisms for controlled nanocarrier delivery.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Nanoestruturas/química , Animais , Portadores de Fármacos/química , Feminino , Camundongos , Micelas , Polímeros/química
9.
Sci Rep ; 5: 9830, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25996055

RESUMO

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.

11.
Sci Rep ; 4: 6854, 2014 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-25358460

RESUMO

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.


Assuntos
Magnetospirillum/citologia , Magnetospirillum/ultraestrutura , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência
12.
Ultramicroscopy ; 110(7): 866-76, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20447768

RESUMO

Spatially resolved low-loss electron energy-loss spectroscopy (EELS) is a powerful method to quantitatively determine the water distribution in frozen-hydrated biological materials at high spatial resolution. However, hydrated tissue, particularly its hydrophilic protein-rich component, is very sensitive to electron radiation. This sensitivity has traditionally limited the achievable spatial resolution because of the relatively high noise associated with low-dose data acquisition. We show that the damage caused by high-dose data acquisition affects the accuracy of a multiple-least-squares (MLS) compositional analysis because of inaccuracies in the reference spectrum used to represent the protein. Higher spatial resolution combined with more accurate compositional analysis can be achieved if a reference spectrum is used that better represents the electron-beam-damaged protein component under frozen-hydrated conditions rather than one separately collected from dry protein under low-dose conditions. We thus introduce a method to extract the best-fitting protein reference spectrum from an experimental spectrum dataset. This method can be used when the MLS-fitting problem is sufficiently constrained so that the only unknown is the reference spectrum for the protein component. We apply this approach to map the distribution of water in cryo-sections obtained from frozen-hydrated tissue of porcine skin. The raw spectral data were collected at doses up to 10(5)e/nm(2) despite the fact that observable damage begins at doses as low as 10(3)e/nm(2). The resulting spatial resolution of 10nm is 5-10 times better than that in previous studies of frozen-hydrated tissue and is sufficient to resolve sub-cellular water fluctuations as well as the inter-cellular lipid-rich regions of skin where water-mediated processes are believed to play a significant role in the phenotype of keratinocytes in the stratum corneum.


Assuntos
Água Corporal/química , Pele/química , Espectroscopia de Perda de Energia de Elétrons/métodos , Algoritmos , Animais , Criopreservação , Técnicas In Vitro , Análise dos Mínimos Quadrados , Nanotecnologia , Proteínas/química , Pele/efeitos da radiação , Pele/ultraestrutura , Espectroscopia de Perda de Energia de Elétrons/estatística & dados numéricos , Suínos
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