Cytosolic Uptake of Large Monofunctionalized Dextrans.
ABSTRACT: Dextrans are a versatile class of polysaccharides with applications that span medicine, cell biology, food science, and consumer goods. Here, we report on a new type of large monofunctionalized dextran that exhibits unusual properties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell types without the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake occurs primarily through active transport via receptor-mediated processes. These monofunctionalized dextrans could serve as intracellular delivery platforms for drugs or other cargos.
Project description:The volumes from which 3H-labelled dextrans are excluded by dermal collagenous fibres were calculated by dilution of dextran probes. Five dextrans, of average Stokes' radii 1.72, 2.53, 3.92, 4.54 and 14.24nm, were investigated at concentrations between 0.1 and 3% (w/w). The excluded volume was dependent on dextran concentration only for the two smaller probes. The largest dextran was shown not to bind to the fibres. A plot of the square root of excluded volume against Stokes' radius was linear for the four smallest dextrans, corresponding to the predictions of Ogston's [(1958) Trans. Faraday Soc. 54, 1754--1757] rod-and-sphere model of fibrous exclusion, and suggesting that dextrans of Stokes' radius between 1.72 and 4.54 nm were excluded by a cylindrical solid fibre of radius 2.90 +/- 0.72 nm. Larger molecules were excluded by a structure of much greater size, since the volume exclusion for the largest dextran was only slightly greater than that of the dextran less than one-third its radius. The excluded volume of 3H2O fell slightly below the line describing the dextran data, indicating that water had access to most of the volume not occupied by the collagenous fibres.
Project description:Insoluble collagen from human dermis was equilibrated in a physiological medium with mixtures of 3H2O and fluorescein-conjugated dextrans of different molecular weights. Dextrans of mol.wts. greater than 10(5) were excluded from a volume of 3.82+/-0.87 ml(S.D.) per g of collagen; dextrans of lower molecular weight occupied a larger volume. The apparent excluded volume was proportional to the weight of the collagen. Dansylated albumin behaved similarly to dextran; the polymeric collagen from rat skin exhibited a much larger excluded volume than the insoluble collagen. These results indicated that the volume available to the plasma proteins in human dermis was limited by insoluble collagen as well as by the glycosaminoglycans of the tissue.
Project description:Novel (S)-camptothecin-dextran polymers were obtained by "click" grafting of azide-modified (S)-camptothecin and alkyne-modified dextrans. Two series based on 10 kDa and 70 kDa dextrans were prepared with a degree of substitution of (S)-camptothecin between 3.1 and 10.2%. The binding properties with ?-cyclodextrin and ?-cyclodextrin polymers were measured by isothermal titration calorimetry and fluorescence spectroscopy, showing no binding with ?-cyclodextrin but high binding with ?-cyclodextrin polymers. In aqueous solution nanoparticles were formed from association between the (S)-camptothecin-dextran polymers and the ?-cyclodextrin polymers.
Project description:The surface coating of solid substrates using dextrans has gained a great deal of attention, because dextran-coated surfaces show excellent anti-fouling property as well as biocompatibility behavior. Much effort has been made to develop efficient methods for grafting dextrans on solid surfaces. This led to the development of catechol-conjugated dextrans (Dex-C) which can adhere to a number of solid surfaces, inspired by the underwater adhesion behavior of marine mussels. The present study is a systematic investigation of the characteristics of surface coatings developed with Dex-C. Various Dex-C with different catechol contents were synthesized and used as a surface coating material. The effect of catechol content on surface coating and antiplatelet performance was investigated.
Project description:Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as <i>O</i>3- and <i>O</i>4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from <i>Bacteroides thetaiotaomicron</i> and <i>Streptococcus salivarius</i> were cloned and heterologously expressed in <i>Escherichia coli</i>. The two recombinant enzymes as well as two commercial dextranases from <i>Chaetomium</i> sp. and <i>Penicillium</i> sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from <i>Chaetomium</i> sp., <i>Penicillium</i> sp., and <i>Bacteroides thetaiotaomicron</i>, isomaltose was the end product of the hydrolysis from linear dextrans, whereas <i>Penicillium</i> sp. dextranase led to isomaltose and isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when incubated with isomaltotriose. For <i>O</i>3- and <i>O</i>4-branched dextrans, the fungal dextranases yielded significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity.
Project description:Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B<sub>2</sub>). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their <i>rrs</i> and <i>pheS</i> gene sequences as LAB belonging to four species (<i>Weissella cibaria</i>, <i>Leuconostoc citreum</i>, <i>Leuconostoc falkenbergense</i> and <i>Leuconostoc mesenteroides</i>). Six selected strains from the <i>Leuconostoc</i> (3) and <i>Weissella</i> (3) genera grew in the absence of riboflavin and synthesized vitamin B<sub>2</sub>. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the <i>L. citreum</i> polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the <i>W. cibaria</i> strains produce the highest levels (585-685 μg/and 6.5-7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by <i>L. falkenbergense</i>.
Project description:The effect of dextrans of various molecular weights on the solubility of fibrinogen was investigated. The results indicate that the prime effect responsible for the observed changes of solubility was steric exclusion. This applies over the whole molecular-weight range of dextrans used, i.e. from 5700 to 76000.
Project description:Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be realized by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real-time imaging of three different endocytic ligands-folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single-particle tracking of Gal3- or STxB-functionalized QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs, which bear a unique stoichiometry of endocytic ligands, represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics.
Project description:Nanoparticle physicochemical properties such as surface charge are considered to play an important role in cellular uptake and particle-cell interactions. In order to systematically evaluate the role of surface charge on the uptake of iron oxide nanoparticles, we prepared carboxymethyl-substituted dextrans with different degrees of substitution, ranging from 38 to 5 groups per chain, and reacted them using carbodiimide chemistry with amine-silane-coated iron oxide nanoparticles with narrow size distributions in the range of 33-45 nm. Surface charge of carboxymethyl-substituted dextran-coated nano-particles ranged from -50 to 5 mV as determined by zeta potential measurements, and was dependent on the number of carboxymethyl groups incorporated in the dextran chains. Nanoparticles were incubated with CaCo-2 human colon cancer cells. Nanoparticle-cell interactions were observed by confocal laser scanning microscopy and uptake was quantified by elemental analysis using inductively coupled plasma mass spectroscopy. Mechanisms of internalization were inferred using pharmacological inhibitors for fluid-phase, clathrin-mediated, and caveola-mediated endocytosis. Results showed increased uptake for nanoparticles with greater negative charge. Internalization patterns suggest that uptake of the most negatively charged particles occurs via non-specific interactions.