Bjørn T. Stokke

Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers

Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow dissociation and release of plasmid DNA, resulting in a slow onset of action. We therefore developed highly effective nonviral gene delivery systems with improved physical properties from a series of chitosan oligomers, ranging in molecular weight from 1.2 to 10 kDa. First, we established structure–property relationships with regard to polyplex formation and in vivo efficiency after lung administration to mice. In a second step, we isolated chitosan oligomers from a preferred oligomer fraction to obtain fractions, ranging from 10 to 50-mers, of more homogeneous size distributions with polydispersities ranging from 1.01 to 1.09. Polyplexes based on chitosan oligomers dissociated more easily than those of a high-molecular-weight ultrapure chitosan (UPC, approximately a 1000-mer), and released pDNA in the presence of anionic heparin. The more easily dissociated polyplexes mediated a faster onset of action and gave a higher gene expression both in 293 cells in vitro and after lung administration in vivo as compared to the more stable UPC polyplexes. Already 24 h after intratracheal administration, a 120- to 260-fold higher luciferase gene expression was observed compared to UPC in the mouse lung in vivo. The gene expression in the lung was comparable to that of PEI (respective AUCs of 2756±710 and 3320±871 pg luciferase × days/mg of total lung protein). In conclusion, a major improvement of chitosan-mediated nonviral gene delivery to the lung was obtained by using polyplexes of well-defined chitosan oligomers. Polyplexes of oligomer fractions also had superior physicochemical properties to commonly used high-molecular-weight UPC.

The cytokine stimulating activity of (1→3)-β-d-glucans is dependent on the triple helix conformation

The immunomodulating properties of comb-like branched (1-->3)-beta-D-glucans scleroglucan, schizophyllan and lentinan depend on branching pattern, molecular weight and higher-order structure. The effect of weight average molecular weight Mw and higher order structure of scleroglucan, on stimulation of human monocytes cultured in vitro to secrete tumor necrosis factor-alpha (TNF-alpha) was investigated. The higher order structures of the scleroglucan samples were determined by electron microscopy. The data showed that the samples with a linear wormlike, triple helical structure with Mw less than 50 x 10(4) g/mol or larger than 110 x 10(4) g/mol stimulated the monocytes more efficiently than samples with Mw in the range (67-110) x 10(4) g/mol. The denaturation of the linear triple helices by NaOH (> 0.25 M), followed by neutralization yielded blends of linear and macrocyclic topologies with concomitant irreversible reduction of the cytokine inducing activity compared with the untreated scleroglucans. The dose-dependent ability to activate monocytes to cytokine production was not restored following annealing of the denatured-renatured samples, despite the fact that electron micrographs revealed similar structures of these annealed samples to the starting material. Pre-incubation of monocytes with antibodies against cluster of differentiation antigens CD14 or CD11b reduced the scleroglucan potency to stimulate TNF-alpha secretion mainly for mAb against CD14 in the presence of serum.

Determination of Glucose Levels Using a Functionalized Hydrogel−Optical Fiber Biosensor: Toward Continuous Monitoring of Blood Glucose in Vivo

Glucose-selective optical sensors were fabricated by incorporating 3-phenylboronic acid and a tertiary amine, dimethylaminopropylacrylamide, into a hydrogel matrix. Determination of glucose in solution is based on the glucose-induced contraction of the hydrogel. The gel was fabricated on the end of an optical fiber, and the optical length was measured by an interferometric technique. Previously it was found the gel could be tuned for enhanced glucose sensitivity and selectivity by varying the 3-phenylboronic acid/tertiary amine ratio. The properties of the responsive hydrogel as a glucose sensor were determined in more detail with respect to swelling kinetics and equilibrium swelling degree. Temperature effects, size variation, molecular interference, and reversibility were addressed. Results showed there was a good degree of reversibility, both for equilibrium swelling and swelling kinetics. Fabricated hydrogel sensors with slight differences in size yielded an overlapping relative response indicating an excellent degree of sensor reproducibility. The sensor proved to be temperature-dependent; by increasing the temperature from 25 to 37 degrees C, the swelling was about 4-fold more rapid, and a concomitant decrease in equilibrium swelling was seen. Identified interference from other analytes with determination of glucose was used a basis for selecting ethylenediaminetetraacetic acid (EDTA) as an anticoagulant for in vitro determination of glucose concentration in blood plasma. Glucose measurements performed in blood plasma were promising, showing that the sensor is capable of measuring physiological glucose levels in blood with a minimal effect from interfering molecules. The obtained results indicate that the developed sensor is a candidate for continuous monitoring of glucose in blood.

Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans

A new method for determining the specificity of hydrolysis of the linear binary heteropolysaccharide chitosan composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN; D-unit) residues is described. The method is based on the assignments of the 13C chemical shifts of the identity (A- or D-units) of the new reducing and non-reducing ends and the variation in their nearest neighbours, using low molecular weight chitosans with known random distribution of A- and D-units as substrate. A highly N-acetylated chitosan with fraction of acetylated units (FA) of 0.68 and a number-average degree of polymerization (DPn) of 30 was hydrolysed with hen egg-white lysozyme, showing that both the new reducing and non-reducing ends consisted exclusively of A-units, indicating a high specificity for A-units in subsites DL and EL on lysozyme. Our data suggests that the preceding unit of the reducing A-units, is invariable, and based on earlier studies, most probably an A-unit, while the unit following the non-reducing A-units can be either an A- or a D-unit. A more detailed study of the specificity of lysozyme at subsite DL was performed by hydrolyzing a more deacetylated chitosan (FA = 0.35 and DPn of 20) to a DPn of 9, showing that even for this chitosan more than 90% of the new reducing ends were acetylated units. Thus, lysozyme depolymerizes partially N-acetylated chitosans by preferentially hydrolyzing sequences of acetylated units bound to site CL, DL and EL of the active cleft, while there is no specificity between acetylated and deacetylated units to site FL. In addition, a moderately N-acetylated chitosan with fraction of acetylated units (FA) of 0.35 and a DPn of 20 was hydrolysed with Bacillus sp. No. 7-M chitosanase, showing that both the new reducing and non-reducing ends consisted exclusively of D-units. Our data suggests that the nearest neigbour to the D-unit at the reducing end is invariable, and based on earlier studies, most probably a D-unit, while the unit following the non-reducing D-units can be either an A- or a D-unit. We conclude that the Bacillus chitosanase hydrolyzes partially N-acetylated chitosan by preferentially attacking sequences of three consecutive deacetylated units, hypothetical subsites CC, DC and EC, where the cleavage occur between sugar units bound to subsites DC and EC. A hypothetical subsite FC on the chitosanase show no specificity with respect to A- and D-units. The new NMR method described herein offers a time and labour-saving alternative to the procedure of extensive hydrolysis of the binary heteropolysaccharide chitosan and subsequent isolation and characterization of the oligosaccharides.

Higher order structure of (1,3)-β-D-glucans and its influence on their biological activities and complexation abilities

(1,3)‐β‐D‐Glucans form a group of biologically active biopolymers that exist in different structural organizations depending on the environmental conditions. The biological effect of (1,3)‐β‐D‐glucans is a core issue stimulating large research efforts of the molecular properties and their consequences for action as biological response modifiers. The fascination for these molecules increased further following the finding of their ability to form complexes of defined geometry with a number of structures, ranging from linear architectures as polymers or carbon nanotubes, to globular structures as gold particles or dye molecules. The fascinating information concerning the relationship between sample treatment history and molecular organization has not yet reached out to all the contributors within the field, resulting in unnecessary apparent inconsistencies in the literature. In addition to environmental conditions, the sample history is known to influence on the precise structural organization of these molecules. The present knowledge related to the structure of native as well as denatured, renatured and annealed (1,3)‐β‐D‐glucans is reviewed. The influence of their structural organization on the biological activity and complexation abilities is discussed, and some factors hindering progress in the understanding of their biological effects or complexation abilities are pointed out.

An antitumor, branched (1 → 3)-β-d-glucan from a water extract of fruiting bodies of Cryptoporus volvatus

A water-soluble, (1-->6)-branched (1-->3)-beta-D-glucan (H-3-B) was isolated from a hot-water extract of the fruiting bodies of the fungus, Cryptoporus volvatus (Basidiomycetes). Enzymatic analysis using exo-(1-->3)-beta-D-glucanase and methylation analysis indicated that this polysaccharide has a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, and single, beta-(1-->6)-linked D-glucopyranosyl residues attached as side chains to, on average, every fourth sugar residue of the main chain. This structure was confirmed by 13C NMR spectra of the glucan in Me2SO-d6. The weight-average molecular weight (Mw) of H-3-B was determined to be 44.0 x 10(4) by gel permeation chromatography equipped with a low-angle laser-light-scattering photometer. The electron microscopic observations showed that H-3-B and its sonicated sample (S-H-3-B, Mw = 13.7 x 10(4)) can be described as linear worm-like chains. The mass per unit length for native and sonicated H-3-B was determined to be 1750 and 1780 g mol-1 nm-1, respectively, from the contour lengths obtained by electron microscopy and the molecular weights. These values are in good agreement with that expected for the triple stranded structure. A sample denatured in 0.1 M NaOH and subsequently renatured by neutralization showed a mixture of linear and cyclic structures, and larger aggregates with less well-defined morphology. The H-3-B and S-H-3-B had antitumor activity against the Sarcoma 180 tumor.

The molecular size and shape of xanthan, xylinan, bronchial mucin, alginate, and amylose as revealed by electron microscopy

Electron microscopy of some selected, vacuum-dried and rotary-shadowed, polyelectrolytic polysaccharides and glycoproteins adsorbed to mica indicates that this technique can yield reliable information about polymer conformation for chains with persistence lengths q exceeding about 10 nm. Statistical analyses of the local polymer tangent-direction yield q = 150 nm for double-stranded xanthan, q = 60 nm for single-stranded xanthan, q = 45 nm for xylinan, q = 16 nm for alginate (90% beta-D-mannuronic acid), and q = 15 nm for human-bronchial mucin. These values are all in adequate agreement with values of q obtained by using other techniques. Amylose, on the other hand, appears as non-randomly aligned chains. The observed contour lengths of amylose indicate a mass per unit length of 1440 dalton/nm, consistent with a pseudo-helical conformation.

Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications.

The Recombinant Azotobacter vinelandii Mannuronan C-5-Epimerase AlgE4 Epimerizes Alginate by a Nonrandom Attack Mechanism

The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family ofAzotobacter vinelandii enzymes catalyzing the polymer level epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of the M and G residues (MG blocks). AlgE4 was purified after intracellular overexpression in Escherichia coli, and the activity was shown to be optimal at pH values between 6.5 and 7.0, in the presence of 1–3 mm Ca2+, and at temperatures near 37 °C. Sr2+ was found to substitute reasonably well for Ca2+ in activation, whereas Zn2+ strongly inhibited the activity. During epimerization of alginate, the fraction of GMG blocks increased linearly as a function of the total fraction of G residues and comparably much faster than that of MMG blocks. These experimental data could not be accounted for by a random attack mechanism, suggesting that the enzyme either slides along the alginate chain during catalysis or recognizes a pre-existing G residue as a preferred substrate in its consecutive attacks.

Glucose sensors based on a responsive gel incorporated as a Fabry-Perot cavity on a fiber-optic readout platform

An optical sensor for detection of glucose is implemented by incorporating a carbohydrate sensitive hydrogel as a Fabry-Perot cavity at the end of optical fiber for high sensitivity readout of the gel length. The glucose sensing functionality was achieved by incorporating boronic acid moieties into an acrylamide-based hydrogel. The interaction between glucose and boronic acid changes the driving forces for gel swelling thus inducing a glucose sensitive hydrogel swelling. The effects on the carbohydrate swelling response, with respect to sensitivity and selectivity, by incorporation of a cationic monomer, dimethyl-aminopropyl acrylamide, into the boronic acid functionalized responsive gels were determined. The linear gel swelling response in aqueous solutions at aqueous 2.5mM carbohydrates were determined to -1760nm/mM for glucose whereas mannose, sucrose, fructose and galactose displayed a response of about 10% of the glucose response for the hydrogels containing 10mol% dimethylaminopropyl acrylamide. This gel composition with 10mol% dimethylaminopropyl acrylamide is the most promising for detection of glucose at physiological pH and ionic strength. A mechanism where carbohydrate specific stabilisation of the boronic acid group and possible carbohydrate mediated additional crosslinking of the elastically active polymer chains is suggested.