We propose a label-free biosensor concept based on the charge state manipulation of nitrogen-vacancy (NV) quantum color centers in diamond, combined with an electrochemical microfluidic flow cell sensor, constructed on boron-doped diamond. This device can be set at a defined electrochemical potential, locking onto the particular chemical reaction, whilst the NV center provides the sensing function. The NV charge state occupation is initially prepared by applying a bias voltage on a gate electrode and then subsequently altered by exposure to detected charged molecules. We demonstrate the functionality of the device by performing label-free optical detection of DNA molecules. In this experiment, a monolayer of strongly cationic charged polymer polyethylenimine is used to shift the charge state of near surface NV centers from negatively charged NV- to neutral NV0 or dark positively charged NV+. Immobilization of negatively charged DNA molecules on the surface of the sensor restores the NV centers charge state back to the negatively charged NV-, which is detected using confocal photoluminescence microscopy. Biochemical reactions in the microfluidic channel are characterized by electrochemical impedance spectroscopy. The use of the developed electrochemical device can also be extended to nuclear magnetic resonance spin sensing.
The use of noble metal nanoparticles in biomedical and biotechnological applications is nowadays well established. Particularly, silver nanoparticles (AgNPs) were proven to be effective for instance as a biocide agent. They also find applications in tumor therapies and sensing applications being encouraging tools for in-vivo imaging. In this framework, whenever they are in contact with living systems, they are rapidly coated by a protein corona thereby influencing a variety of biological events including cellular uptake, blood circulation lifetime, cytotoxicity and, ultimately, the therapeutic effect. Taking these considerations into account, we have explored the behavior of polymer-coated AgNPs in model protein environments focusing on the self-development of protein coronas. The polymers polyethyleneimine (PEI), polyvinylpyrrolidone (PVP) and poly(2-vinyl pyridine)-b-poly(ethylene oxide) (PEO-b-P2VP) were used as stabilizing agents. The chemical nature of the polymer capping remarkably influences the behavior of the hybrid nanomaterials in protein environments. The PEO-b-P2VP and PVP-stabilized AgNPs are essentially inert to the model proteins adsorption. On the other hand, the PEI-stabilized AgNPs interact strongly with bovine serum albumin (BSA). Nevertheless, the same silver colloids were evidenced to be stable in IgG and lysozyme environments. The BSA adsorption into the PEI-stabilized AgNPs is most probably driven by hydrogen bonding and van der Waals interactions as suggested by isothermal titration calorimetry data. The development of protein coronas around the AgNPs may have relevant implications in a variety of biological events. Therefore, further investigations are currently underway to evaluate the influence of its presence on the cytotoxicity, hemolytic effects and biocide properties of the produced hybrid nanomaterials.
- MeSH
- adsorpce MeSH
- dynamický rozptyl světla MeSH
- fluorescenční spektrometrie MeSH
- kalorimetrie MeSH
- koloidy chemie MeSH
- kur domácí MeSH
- nanočástice ultrastruktura MeSH
- polyethylenimin chemie MeSH
- polymery chemie MeSH
- povidon chemie MeSH
- proteinová korona chemie MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- spektrofotometrie ultrafialová MeSH
- stříbro chemie MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.
- MeSH
- chemické jevy MeSH
- CRISPR-Cas systémy * MeSH
- editace genu * MeSH
- exprese genu MeSH
- fluorescenční protilátková technika MeSH
- HEK293 buňky MeSH
- koloidy MeSH
- lidé MeSH
- magnetické nanočástice * chemie ultrastruktura MeSH
- plazmidy genetika MeSH
- polyethylenimin * chemie MeSH
- reportérové geny MeSH
- statická elektřina MeSH
- technika přenosu genů * MeSH
- transfekce metody MeSH
- velikost částic MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Herein, we describe a new method for the synthesis of superhydrophilic poly(2-alkyl-2-oxazoline)s (PAOx) from poly(2-ethyl-2-oxazoline) (PEtOx). A well-defined linear polyethylenimine was prepared from PEtOx by controlled acidic hydrolysis of its side-chains followed by reacylation with different carboxylic acids. Using this protocol, we obtained a series of new hydrophilic PAOx containing side-chain ether groups with potential in biomaterials science. The relative hydrophilicity of the polymers was assessed, revealing that poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) is the most hydrophilic PAOx reported to date. Additionally, the amorphous poly(2-methoxy-ethoxy-ethoxymethyl-2-oxazoline) (PDEGOx) shows the lowest reported glass transition temperature (-25 °C) within the PAOx family to date. The biomedical potential of the prepared polymers was further fortified by an in vitro cytotoxicity study, where all polymers appeared to be noncytotoxic. The described synthetic protocol is universal and can be extremely versatile, especially for PAOx that are difficult to prepare by conventional cationic ring-opening polymerization due to the monomer interference and/or degradation.
- MeSH
- HeLa buňky MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- oxazoly chemie MeSH
- polyethylenimin chemie MeSH
- tranzitní teplota MeSH
- vitrifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Magnetic resonance imaging using fluorinated contrast agents (19F MRI) enables to achive highcontrast in images due to the negligible fluorine background in living tissues. In this pilot study, we developed new biocompatible, temperature-responsive, and easily synthesized polymeric nanogels containing a sufficient concentration of magnetically equivalent fluorine atoms for 19F MRI purposes. The structure of the nanogels is based on amphiphilic copolymers containing two blocks, a hydrophilic poly[ N-(2-hydroxypropyl)methacrylamide] (PHPMA) or poly(2-methyl-2-oxazoline) (PMeOx) block, and a thermoresponsive poly[ N(2,2difluoroethyl)acrylamide] (PDFEA) block. The thermoresponsive properties of the PDFEA block allow us to control the process of nanogel self-assembly upon its heating in an aqueous solution. Particle size depends on the copolymer composition, and the most promising copolymers with longer thermoresponsive blocks form nanogels of suitable size for angiogenesis imaging or the labeling of cells (approximately 120 nm). The in vitro 19F MRI experiments reveal good sensitivity of the copolymer contrast agents, while the nanogels were proven to be noncytotoxic for several cell lines.
- MeSH
- fluor chemie MeSH
- HeLa buňky MeSH
- hemolýza účinky léků MeSH
- kontrastní látky škodlivé účinky chemie MeSH
- kultivované buňky MeSH
- kyseliny polymethakrylové chemie MeSH
- lidé MeSH
- magnetická rezonanční tomografie metody MeSH
- myši MeSH
- polyaminy chemie MeSH
- polyethylenglykoly chemie MeSH
- polyethylenimin chemie MeSH
- polymerizace MeSH
- teplota MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Synthesis of theranostic nanoparticles, which combine both therapeutic and diagnostic capabilities in one platform can be considered as a step forward personalized medicine, since it allows tracing the delivery of the drug to targeted organ. Thus, the aim of this work was to prepare gadolinium alginate gel nanoparticles (gadolinum nanogels - GdNG) by the reverse microemulsions and physical crosslinking method as the vehicles able to carry hydrophilic drugs and to be traced by the Magnetic Resonance Imaging (MRI). The average size of synthesized nanoparticles was about 110nm and the batch concentration was 10(10) particles/ml. The morphology of nanogeles was visualized by Cryo-Scanning Electron Microscopy. Surface of nanogels particles was modified by the Layer-by-Layer (LbL) technique using natural polyelectrolytes. The cytotoxicity of non-modified and LbL modified nanogels was evaluated by the cellular viability quantification and cell death assessments using MTT and LDH biochemical tests, respectively. We encapsulated the model compound - fluorescent dye (Rhodamine b) in nanogels networks and proved the possibility of GdNG visualization by MRI.
- MeSH
- algináty chemie MeSH
- buněčná smrt MeSH
- fluorescenční barviva chemie MeSH
- gadolinium chemie MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- nádorové buněčné linie MeSH
- polyethylenglykoly chemická syntéza chemie MeSH
- polyethylenimin chemická syntéza chemie MeSH
- teranostická nanomedicína * MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Multifunctional nanoparticles for magnetic hyperthermia which simultaneously display antibacterial properties promise to decrease bacterial infections co-localized with cancers. Current methods synthesize such particles by multi-step procedures, and systematic comparisons of antibacterial properties between coatings, as well as measurements of specific absorption rate (SAR) during magnetic hyperthermia are lacking. Here we report the novel simple method for synthesis of magnetic nanoparticles with shells of oleic acid (OA), polyethyleneimine (PEI) and polyethyleneimine-methyl cellulose (PEI-mC). We compare their antibacterial properties against single gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria as well as biofilms. Magnetite nanoparticles (MNPs) with PEI-methyl cellulose were found to be most effective against both S. aureus and E. coli with concentration for 10% growth inhibition (EC10) of <150 mg/l. All the particles have high SAR and are effective for heat-generation in alternating magnetic fields.
- MeSH
- antibakteriální látky chemie farmakologie MeSH
- biofilmy účinky léků MeSH
- Escherichia coli účinky léků fyziologie MeSH
- indukovaná hypertermie MeSH
- magnetické nanočástice chemie MeSH
- methylcelulosa chemie MeSH
- polyethylenimin chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- Staphylococcus aureus účinky léků fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.
- MeSH
- adenosintrifosfát metabolismus MeSH
- antioxidancia metabolismus farmakologie MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buněčné dýchání účinky léků MeSH
- buněčné linie MeSH
- energetický metabolismus účinky léků MeSH
- glutathion metabolismus MeSH
- homeostáza MeSH
- kinetika MeSH
- lidé MeSH
- mitochondriální membrány účinky léků metabolismus MeSH
- molekulární struktura MeSH
- molekulová hmotnost MeSH
- oxidace-redukce MeSH
- oxidační stres účinky léků MeSH
- polyethylenimin chemie toxicita MeSH
- reaktivní formy kyslíku metabolismus MeSH
- spotřeba kyslíku účinky léků MeSH
- transfekce metody MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.
- MeSH
- buňky NK metabolismus MeSH
- disulfidy metabolismus MeSH
- DNA metabolismus MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- krystalizace MeSH
- lektiny typu C chemie izolace a purifikace metabolismus MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- N-acetylglukosaminyltransferasy metabolismus MeSH
- polyethylenimin chemie MeSH
- polysacharidy metabolismus MeSH
- rozpustnost MeSH
- roztoky MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- stabilita proteinů MeSH
- terciární struktura proteinů MeSH
- transfekce metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Magnetic harvesting of microalgal biomass provides an attractive alternative to conventional methods. The approach to this issue has so far been pragmatic, focused mainly on finding cheap magnetic agents in combination with harvestable microalgae species. The aim of this work was to study experimentally and theoretically the mechanisms leading to cell-magnetic agent attachment/detachment using real experiments and predictions made by colloidal adhesion (XDLVO) model. Two types of well defined magnetic beads (MBs) carrying ion exchange functional groups (DEAE - diethylaminoethyl and PEI - polyethylenimine) were studied in connection with microalgae (Chlorella vulgaris). Optimal harvesting efficiencies (>90%) were found for DEAE and PEI MBs, while efficient detachment was achieved only for DEAE MBs (>90%). These findings were in accordance with the predictions by XDLVO model. Simultaneously there was found a discrepancy between the XDLVO prediction and the poor detachment of PEI MBs from microalgal surface. This can be ascribed to an additional interaction (probably covalent bonds) between PEI and algal surface, which the XDLVO model is unable to capture given by its non-covalent nature.
- MeSH
- biologické modely MeSH
- biomasa MeSH
- buněčná adheze MeSH
- Chlorella vulgaris izolace a purifikace fyziologie MeSH
- ethanolaminy chemie MeSH
- iontová výměna MeSH
- koloidy MeSH
- magnetické jevy MeSH
- magnetické nanočástice chemie MeSH
- mikrořasy izolace a purifikace fyziologie MeSH
- polyethylenimin chemie MeSH
- povrchové vlastnosti MeSH
- průmyslová mikrobiologie metody MeSH
- sladká voda mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
