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1.
MethodsX ; 6: 212-218, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30766801

RESUMO

Polymeric amines and complex amine containing system are actively studied and applied as gene delivery agents in gene therapy and genetic engineering. Optimizing polymer - nucleic acid ratio is the key stage in elaboration of procedures in this area. Application of fluorescent tagged oligonucleotides is widespread approach which allows to visualize nucleic acid in gel electrophoresis experiments and to find conditions of the full binding of the nucleic acid. We suggest to use succinimidyl ester of 7-(diethylamino)coumarin-3-carboxylic acid as an agent for fluorescent labeling of polymeric amines and to use the tagged polymers in optimizing polymer - nucleic acid ratio. This approach allows to see unbound polymer and to study various nucleic acids in interaction with the same polymer. •Labeling of gene delivery agents with fluorescence groups increases efficiency of optimization of gene delivery compositions.•Polymeric amines tagged with succinimidyl ester of 7-(diethylamino)coumarin-3-carboxylic acid are suitable for study polymer - nucleic acid interaction with gel electrophoresis.

2.
Plant Physiol Biochem ; 125: 205-211, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29475086

RESUMO

Siliceous frustules of diatom algae contain unique long-chain polyamines, including those having more than six nitrogen atoms. These polyamines participate in the formation of the siliceous frustules of the diatoms but their precise physiological role is not clear. The main hypotheses include formation of a polyamine and polyphosphate supramolecular matrix. We have synthesized novel fluorescent dyes from a synthetic oligomeric mixture of polyamines and the fluorophore 7-nitro-2,1,3-benzoxadiazole. The long polyamine chain ensures the high affinity of these dyes to silica, which allows their application in the staining of siliceous materials, such as valves of diatom algae and fossilized samples from sediments. The fluorescently stained diatom valves were found to be promising liquid flow tracers in hydrodynamic tests. Furthermore, complexation of the polyamine component of the dyes with carbonic polymeric acids results in changes to the visible spectrum of the fluorophore, which allows study of the stability of the complex vs the length of the polyamine chain. Using poly (vinyl phosphonic acid) as a model for phosphate functionality in silaffins (a potential matrix in the formation of biogenic silica) little complexation with the polyamine fluorophores was observed, bringing into question the role of a polyamine - polymeric phosphate matrix in biosilicification.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Poliaminas Biogênicas , Diatomáceas , Corantes Fluorescentes/química , Coloração e Rotulagem/métodos , Poliaminas Biogênicas/química , Poliaminas Biogênicas/metabolismo , Diatomáceas/citologia , Diatomáceas/metabolismo
3.
Polymers (Basel) ; 10(12)2018 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-30961222

RESUMO

Amine containing polymers are extensively studied as special carriers for short-chain RNA (13⁻25 nucleotides), which are applied as gene silencing agents in gene therapy of various diseases including cancer. Elaboration of the oligonucleotide carriers requires knowledge about peculiarities of the oligonucleotide⁻polymeric amine interaction. The critical length of the interacting chains is an important parameter which allows us to design sophisticated constructions containing oligonucleotide binding segments, solubilizing, protective and aiming parts. We studied interactions of (TCAG)n, n = 1⁻6 DNA oligonucleotides with polyethylenimine and poly(N-(3-((3-(dimethylamino)propyl)(methyl)amino)propyl)-N-methylacrylamide). The critical length for oligonucleotides in interaction with polymeric amines is 8⁻12 units and complexation at these length can be accompanied by "all-or-nothing" effects. New dimethylacrylamide based polymers with grafted polyamine chains were obtained and studied in complexation with DNA and RNA oligonucleotides. The most effective interaction and transfection activity into A549 cancer cells and silencing efficiency against vascular endothelial growth factor (VEGF) was found for a sample with average number of nitrogens in polyamine chain equal to 27, i.e., for a sample in which all grafted chains are longer than the critical length for polymeric amine⁻oligonucleotide complexation.

4.
Polymers (Basel) ; 9(11)2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30965927

RESUMO

Polymeric amines are intensively studied due to various valuable properties. This study describes the synthesis of new polymeric amines and ampholytes by the reaction of poly(acryloyl chloride) with trimethylene-based polyamines containing one secondary and several (1⁻3) tertiary amine groups. The polymers contain polyamine side chains and carboxylic groups when the polyamine was in deficiency. These polymers differ in structure of side groups, but they are identical in polymerization degree and polydispersity, which facilitates the study of composition-properties relationships. The structure of the obtained polymers was confirmed with 13C nuclear magnetic resonance infrared spectroscopy, and acid-base properties were studied with potentiometry titration. Placement of the amine groups in the side chains influences their acid-base properties: protonation of the amine group exerts a larger impact on the amine in the same side chain than on the amines in the neighboring side chains. The obtained polymers are prone to aggregation in aqueous solutions tending to insolubility at definite pH values in the case of polyampholytes. Silicic acid condensation in the presence of new polymers results in soluble composite nanoparticles and composite materials which consist of ordered submicrometer particles according to dynamic light scattering and electron microscopy. Polymeric amines, ampholytes, and composite nanoparticles are capable of interacting with oligonucleotides, giving rise to complexes that hold promise for gene delivery applications.

5.
J Struct Biol ; 194(1): 29-37, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26821342

RESUMO

Siliceous sponges are the most primitive multicellular animals whose skeleton consists of spicules - needle-like constructions from silicon dioxide surrounding organic axial filaments. Mechanisms of spicule formation have been intensively studied due to the high ecological importance of sponges and their interest to materials science. Light and electron microscopy are not appropriate enough to display the process from silicon-enriched cells to mature spicules because of composite structure of the sponge tissues. In this article, spiculogenesis in the siliceous sponge has been studied for the first time with the use of fluorescent microscopy. Fluorescent vital dye NBD-N2 was applied to stain growing siliceous structures in the sponge and primmorph cell system. The main stages of spicule growth in the fresh-water sponge Lubomirskia baicalensis (Pallas, 1773) were visualized: silicon accumulation in sclerocytes; formation of an organic filament protruding from the cell; further elongation of the filament and growth of the spicule in a spindle-like form with enlargement in the center; merger with new sclerocytes and formation of the mature spicule. Fluorescent microscopy combined with SEM allows us to overcome the virtual differentiation between intra- and extracellular mechanisms of spicule growth. The growing spicule can capture silicic acid from the extracellular space and merge with new silicon-enriched cells. Visualization of the growing spicules with the fluorescent dye allows us to monitor sponge viability in ecological or toxicological experiments and to apply genomic, proteomic and biochemical techniques.


Assuntos
Corantes Fluorescentes/química , Poríferos/química , Poríferos/citologia , Dióxido de Silício/química , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Animais , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Lagos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Poríferos/ultraestrutura , Propilaminas/química
6.
Anal Biochem ; 486: 5-13, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26103595

RESUMO

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochemistry, as a derivatization agent in analytical chemistry, and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines containing two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compound formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated aromatic systems for amine derivatization in analytical and biochemistry applications. The reaction with polyamines opens the way to libraries of NBD-tagged compounds.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Corantes Fluorescentes/química , Poliaminas/química , 4-Cloro-7-nitrobenzofurazano/síntese química , Animais , Diatomáceas/química , Corantes Fluorescentes/síntese química , Poríferos/química , Coloração e Rotulagem
7.
J Colloid Interface Sci ; 446: 1-10, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25646785

RESUMO

Polymeric amines have been intensively studied for application in smart systems and as matrices for the design of composite materials, including bioinspired substances. A new thermo- and pH-responsive polymer was obtained by radical polymerization of N-(3-(diethylamino)propyl)-N-methylacrylamide. Upon heating, the polymer precipitated from aqueous solutions above pH 9; the observed cloud point was dependent on the polymer concentration and decreased from 95°C at pH 9 to 40°C at pH 11. The basicity of the polymer decreased at elevated temperatures owing to an increase in the hydrophobicity-driven compaction of the macromolecules. Dynamic light scattering analysis demonstrated that the formation of large multimolecular associates with radius 1000-2000 nm was initiated from 1 to 2°C below the cloud point. The new polymer is demonstrated to be an effective matrix for various siliceous composite structures, including 200-300 nm solid spherical raspberry-like particles and hollow hemispherical particles of more than 1000 nm diameter. Condensation of silicic acid in the presence of polymeric amines is a model reaction in biosilicification studies, and the obtained data are also discussed from the perspective of the matrix hypothesis for biosilica formation.


Assuntos
Aminas/química , Substâncias Macromoleculares/química , Nanocompostos/química , Polímeros/química , Dióxido de Silício/química , Água/química , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Propriedades de Superfície , Temperatura Ambiente
8.
Eur Biophys J ; 42(7): 587-90, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23709009

RESUMO

Diatom algae realize highly intriguing processes of biosynthesis of siliceous structures in living cells under moderate conditions. Investigation of diatom physiology is complicated by frustule (siliceous exoskeleton). Frustules consist of valves and girdle bands which are adhered to each other by means of organic substances. Removal of the frustule from the lipid membrane of diatom cells would open new possibilities for study of silicon metabolism in diatoms. We found that submillimeter laser irradiation produced by a free-electron laser causes splitting of diatom frustules without destruction of cell content. This finding opens the way to direct study of diatom cell membrane and to isolation of cell organelles, including silica deposition vesicles. We suppose that the dissection action of the submillimeter irradiation results from unusual ultrasonic waves produced by the short (30-100 ps) but high-power (1 MW) terahertz laser impulses at 5.6 MHz frequency.


Assuntos
Parede Celular/efeitos da radiação , Diatomáceas/citologia , Diatomáceas/efeitos da radiação , Lasers , Membrana Celular/efeitos da radiação , Fatores de Tempo
9.
Protoplasma ; 250(5): 1147-55, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23525742

RESUMO

We studied the growth of the araphid pennate diatom Synedra acus subsp. radians (Kützing) Skabichevskii using a fluorescent dye N(1),N(3)-dimethyl-N(1)-(7-nitro-2,1,3-benzoxadiazol-4-yl)propane-1,3-diamine (NBD-N2), which stains growing siliceous frustules but does not stain other subcellular organelles. We used a clonal culture of S. acus that was synchronized by silicon starvation. Epifluorescence microscopy was performed in two different ways with cells stained by the addition of silicic acid and the dye. Individual cells immobilized on glass were observed during the first 15-20 min following the replenishment of silicic acid after silicon starvation. Alternatively, we examined cells of a batch culture at time intervals during 36 h after the replenishment of silicic acid using fluorescence and confocal microscopy. The addition of silicic acid and NBD-N2 resulted in the rapid (1-2 min) formation of several dozen green fluorescent submicrometer particles (GFSPs) in the cytoplasm, which was accompanied by the accumulation of fluorescent silica inside silica deposition vesicles (SDVs) along their full length. In 5-15 min, GFSPs disappeared from the cytoplasm. Mature siliceous valves were formed within the SDVs during the subsequent 14-16 h. In the next 8-10 h, GFSPs appeared again in the cytoplasm of daughter cells. The data obtained confirm observations about the two-stage mechanism of silicon assimilation, which includes rapid silicon uptake (surge uptake) followed by slow silica deposition. It is likely that the observed GFSPs are silicon transport vesicles, which were first proposed by Schmid and Schulz in (Protoplasma 100:267-288, 1979).


Assuntos
Diatomáceas/metabolismo , Silício/metabolismo , Sequência de Aminoácidos , Microscopia Confocal , Dados de Sequência Molecular , Vesículas Transportadoras/metabolismo
10.
Biomacromolecules ; 12(5): 1772-80, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21469744

RESUMO

The role of polymer (poly(vinylamine)) size (238-11000 units) on silicic acid condensation to yield soluble nanoparticles or composite precipitates has been explored by a combination of light scattering (static and dynamic), laser ablation combined with aerosol spectrometry, IR spectroscopy, and electron microscopy. Soluble nanoparticles or composite precipitates are formed according to the degree of polymerization of the organic polymer and pH. Nanoparticles prepared in the presence of the highest molecular weight polymers have core-shell like structures with dense silica cores. Composite particles formed in the presence of polymers with extent of polymerization below 1000 consist of associates of several polymer-silica nanoparticles. The mechanism of stabilization of the "soluble" silica particles in the tens of nanometer size range involves cooperative interactions with the polymer chains which varies according to chain length and pH. An example of the use of such polymer-poly(silicic acid) nanoparticles in the generation of composite polymeric materials is presented. The results obtained have relevance to the biomimetic design of new composite materials based on silica and polymers and to increasing our understanding of how silica may be manipulated (stored) in the biological environment prior to the formation of stable mineralized structures. We suspect that a similar method of storing silicic acid in an active state is used in silicifying organisms, at least in diatom algae.


Assuntos
Nanopartículas , Polivinil/química , Dióxido de Silício/química , Microscopia Eletrônica de Varredura , Espectrofotometria Infravermelho
11.
Rapid Commun Mass Spectrom ; 25(7): 877-88, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21416524

RESUMO

Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H](+) and multiply [M+nH](n+) charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH](n+) were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Poliaminas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Diatomáceas/química , Sedimentos Geológicos/química
12.
Proc Natl Acad Sci U S A ; 105(16): 5963-8, 2008 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-18420819

RESUMO

Considerable research has been directed toward identifying the mechanisms involved in biosilicification to understand and possibly mimic the process for the production of superior silica-based materials while simultaneously minimizing pollution and energy costs. Molecules isolated from diatoms and, most recently sponges, thought to be key to this process contain polyamines with a propylamine backbone and variable levels of methylation. In a chemical approach to understanding the role of amine (especially propylamine) structures in silicification we have explored three key structural features: (i) the degree of polymerization, (ii) the level of amine methylation, and (iii) the size of the amine chain spacers. In this article, we show that there are two factors critical to their function: the ability of the amines to produce microemulsions and the presence of charged and uncharged amine groups within a molecule, with the latter feature helping to catalyze silicic acid condensation by a proton donor/acceptor mechanism. The understanding of amine-silicate interactions obtained from this study has enabled the controlled preparation of hollow and nonporous siliceous materials under mild conditions (circumneutral pH, room temperature, and in all aqueous systems) possibly compatible with the conditions used by biosystems. The "rules" identified from our study were further used predictively to modulate the activity of a given amine. We believe that the outcomes of the present contribution will form the basis for an approach to controlling the growth of inorganic materials by using tailor-made organic molecules.


Assuntos
Materiais Biomiméticos/química , Diatomáceas/química , Poliaminas/química , Silicatos/química , Emulsões , Interações Hidrofóbicas e Hidrofílicas , Metilação , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Prótons
13.
Bioessays ; 30(4): 328-37, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18348175

RESUMO

Many pigmented heterokonts are able to synthesize elements of their cell walls (the frustules) of dense biogenic silica. These include diatom algae, which occupy a significant place in the biosphere. The siliceous frustules of diatoms have species-specific patterns of surface structures between 10 and a few hundred nanometers. The present review considers possible mechanisms of uptake of silicic acid from the aquatic environment, its transport across the plasmalemma, and intracellular transport and deposition of silica inside the specialized Silica Deposition Vesicle (SDV) where elements of the new frustule are formed. It is proposed that a complex of silicic acid with positively charged proteins silaffins and polypropylamines remains a homogeneous solution during the intracellular transport to SDV, where biogenic silica precipitates. The high density of the deposited biogenic silica may be due to removal of water from the SDV by aquaporins followed by syneresis--a process during which pore water is expelled from the network of the contracting gel. The pattern of aquaporins in the silicalemma, the membrane embracing the SDV, can determine the pattern of species-specific siliceous nanostructures.


Assuntos
Biotecnologia/métodos , Diatomáceas/fisiologia , Nanopartículas/química , Nanotecnologia/métodos , Silício/química , Aquaporinas/química , Química/métodos , Humanos , Modelos Químicos , Pigmentação , Quartzo/química , Ácido Silícico/química
14.
Chem Commun (Camb) ; (14): 1521-3, 2006 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-16575447

RESUMO

A new method for the stepwise synthesis of propylamines containing fragments of N-methyl propylamine as found in diatom bioextracts is presented and their activity in silicic acid condensation is described.


Assuntos
Diatomáceas/química , Peptídeos/química , Propilaminas/síntese química , Precipitação Química , Poliaminas/síntese química , Ácido Silícico/química
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