High Molecular Weight Dextran Research Articles

Characterization and potential applications of high molecular weight dextran produced by Leuconostoc mesenteroides AA1.

Different strains of Leuconostoc mesenteroides were isolated from indigenous sources that were capable of producing a commercially important long chain glucose polymer known as dextran. Dextran produced by L. mesenteroides AA1 was characterized. The physicochemical characteristics and structural analysis of dextran revealed that this biopolymer has many potential applications in several biotechnological industries and L. mesenteroides AA1 can be used for commercial production of dextran on large scale. Dextran produced by this strain is a high molecular weight glucose polymer with only α (1→6) branch linkage in the main backbone chain without any further branching with a viscosity of 2.23×103cp of 5.0% dextran solution. This high molecular weight dextran has an average molecular weight ranging from 10,000 to 40,000kDa. The key utility of dextran in different industries lies on its molecular weight and dextran produced by L. mesenteroides AA1 can be tailored to meet the requirement for any industry.

Purification and characterization of extracellular dextranase from a novel producer, Hypocrea lixii F1002, and its use in oligodextran production

Fungi were isolated from natural soil samples and screened for extracellular dextranase synthesis. The strain F1002 was identified as Hypocrea lixii using a standard internal transcribed spacer ribosomal DNA analysis and was selected for extracellular dextranase synthesis. The enzyme was purified via ammonium sulfate precipitation and Sepharose 6B chromatography, which resulted in an 8.3-fold increase in the specific activity and a 10.73% recovery. This enzyme is a monomeric protein with a molecular mass of 62kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme, which was identified as an endodextranase, had an optimum pH of 5.0 and an optimum temperature of 25°C. The dextranase activity was enhanced by Mg2+, Al3+, and especially Zn2+ at a low concentration, which improved its activity to 124.22%. The enzyme has a very high hydrolytic affinity toward high-molecular weight dextrans. Setting the concentrations of the H. lixii F1002 dextranase (2.31U/mL) and dextrans (6%), as well as the reaction time (45min), allowed the dextranase to hydrolyze dextrans of controlled molecular weights (20–70kDa). Three types of oligodextrans with different molecular weights (namely, 69,376, 38,251, and 21,364Da) were obtained, with a total yield of 80.32%.

High-Resolution, Noninvasive, Two-Photon Fluorescence Measurement of Molecular Concentrations in Corneal Tissue

To perform high-resolution, noninvasive, calibrated measurements of the concentrations and diffusion profiles of fluorescent molecules in the live cornea after topical application to the ocular surface. An 800-nm femtosecond laser was used to perform two-photon fluorescence (TPF) axial scanning measurements. Calibration solutions consisting of sodium fluorescein (Na-Fl; concentration range, 0.01%-2.5%) and riboflavin (concentration range, 0.0125%-0.1%) were tested in well slides, and TPF signals were assessed. Excised feline eyeballs preserved in corneal storage medium and with either intact or removed corneal epithelia were then treated with Na-Fl, riboflavin, or fluorescein dextran (Fl-d) of different molecular weight (MW) for 30 minutes. Calibrated TPF was then used immediately to measure the concentration of these molecules across the central corneal depth. The axial resolution of our TPF system was 6 μm, and a linear relationship was observed between TPF signal and low concentrations of most fluorophores. Intact corneas treated with Na-Fl or riboflavin exhibited a detectable penetration depth of only approximately 20 μm, compared with approximately 400 to 600 μm when the epithelium was removed before fluorophore application. Peak concentrations for intact corneas were half those attained with epithelial removal. Debrided corneas treated with 2,000,000 MW Fl-d showed a half-maximum penetration depth of 156.7 μm compared with 384 μm for the 3,000 MW dextran. The peak concentration of the high MW dextran was one quarter that of the lower MW dextran. TPF is an effective, high-resolution, noninvasive method of quantifying the diffusion and concentration of fluorescent molecules across the cornea.

Targeted Inhibition of Connexin 43 Hemichannels Blunts Ca 2+-Induced Intercellular Dyssynchrony and ATP Efflux in Hl-1 Cardiomyocyte Syncitia

Gap junctions (GJ) are essential conduits that underpin cell-to-cell coupling between cardiomyocytes and are formed from the coalescence of connexin (Cx) hemichannels from two close opposed cells. However, there is evidence supporting a (patho)physiologic role for ‘unpaired’ Cx hemichannels as they traffic through the plasma membrane (PM) to the GJ. We used beating HL-1 cardiomyocyte monolayers as a model system for GJ intercellular communication (GJIC) and to investigate the functional role of Cx hemichannels in syncitial behaviour. GJIC was quantified as an index of intercellular Ca2+ release synchrony and was reconciled with detailed spatio-temporal analysis of intracellular Ca2+ signalling. Ouabain-evoked Ca2+ perturbation inhibited GJIC in a dose-dependent manner and was associated with reduced cell viability. We hypothesised that the intercellular dyssynchrony and cell death linked to ouabain-induced Ca2+ dysfunction was exacerbated by aberrant Cx hemichannel opening that may also compromise cellular metabolism. Consistent with this concept, the magnitude of intracellular Ca2+ flux dysfunction correlated with ATP release from cells. Trans-PM ATP leak was attenuated by Gap20, a peptide corresponding to an intracellular loop motif in Cx43. Moreover, Gap20 reduced intracellular Ca2+ perturbation, improved intercellular synchrony and reduced cell death in ouabain-treated syncitia. The lack of efficacy of a peptide corresponding to a similar epitope in Cx26, and also following the conjugation of Gap20 to a high molecular weight dextran confirmed i) the specificity of this approach and ii) that peptide bioactivity is dependent on its entry into cells and interaction with the intracellular face of Cx. Our data provides evidence that altered cellular Ca2+ homeostasis opens Cx hemichannels and that this may accelerate the metabolic deterioration of cardiomyocytes and exacerbate cardiac dysfunction in situ.

Development and Characterization of a Novel Human In Vitro Blood-Nerve Barrier Model Using Primary Endoneurial Endothelial Cells

There are phenotypic and functional differences between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells (ECs) from the same tissue. Relatively little is known about the human blood-nerve barrier (BNB). We report the development of an in vitro BNB model using primary human endoneurial ECs freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion, and density centrifugation. Primary human endoneurial ECs are spindle shaped and contact inhibited. They rapidly differentiate to form capillary-like networks and microvessels, bind Ulex Europaeus Agglutinin 1 lectin, express von Willebrand factor, and endocytose acetylated low-density lipoprotein. They also express specific transport and cellular adhesion molecules and tight junction proteins, consistent with cells that form a highly restrictive endothelial barrier similar to the blood-brain barrier. When cultured on collagen-coated transwell inserts, the primary human endoneurial ECs develop an in vitro BNB with high transendothelial electrical resistances (160 Omega x cm(2); maximal 12 days after seeding) and low solute permeability coefficient to fluoresceinated high-molecular weight (70 kDa) dextran (2.75 x 10(-3) cm/minute). This in vitro BNB model retains essential known or expected characteristics of the human BNB and has many potential applications for studies of solute, macromolecule, microbial, virus, and leukocyte interactions with this highly specialized endothelial barrier.

Investigation of Genipin Cross-Linked Microcapsule for Oral Delivery of Live Bacterial Cells and Other Biotherapeutics: Preparation and In Vitro Analysis in Simulated Human Gastrointestinal Model

Oral therapy utilizing engineered microorganisms has shown promise in the treatment of many diseases. By microencapsulation, viable cells can overcome the harsh gastrointestinal (GI) environment and secrete needed therapeutics into the gut. These engineered cells should be encased without escaping into the GI tract for safety concerns, thus robust microcapsule membrane is requisite. This paper examined the GI performance of a novel microcapsule membrane using a dynamic simulated human GI model. Results showed that the genipin cross-linked alginate-chitosan (GCAC) microcapsules possessed strong resistance to structural disintegration in the simulated GI environment. Leakage of encapsulated high molecular weight dextran, a model material to be protected during the simulated GI transit, was negligible over 72 h of exposure, in contrast to considerable leakage of dextran from the non-cross-linked counterparts. These microcapsules did not alter the microflora and enzymatic activities in the simulated human colonic media. This study suggested the potential of the GCAC microcapsules for oral delivery of live microorganisms and other biotherapeutics.

Effect of dextran 500 on radial migration of erythrocytes in postcapillary venules at low flow rates.

Recently, we reported that collision efficiency (fraction of total collisions that result in the formation of aggregates) between red blood cells was an important factor in the formation of aggregates in postcapillary venules. In the present study, we focus on how high molecular weight dextran influences the overall radial migration trend of red blood cells in the postcapillary venule along a longitudinal distance of 50 microm from the bifurcation which would in turn affect collision behavior of these cells. A radial migration index, which defines the extent of radial migration of individual cells relative to the vessel center, was found to have a larger magnitude after infusion of dextran (1.9 +/- 2.73) compared to that before dextran infusion (1.48 +/- 3.89). This implied that dextran-induced aggregation might provide an external force to actively move cells towards the centerline of the vessel, which could contribute to the greater number of red blood cells participating in collision (16% increase) and aggregate formation. Further analysis of the collision behavior of individual red blood cells revealed that collision frequencies of individual cells decreased from a wide range (1 to 14) to a narrow range (1 to 5) after dextran treatment, indicating the alteration of collision behavior of red blood cells by the presence of aggregates along the flow stream.

Development of a human in vitro blood-nerve barrier model (94.20)

Abstract Phenotypic and functional differences exist between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells from the same tissue. Little is known about cellular interactions at the blood-nerve barrier (BNB). An in vitro BNB (IVBNB) model was developed using primary human endoneurial endothelial cells (pHEndECs) freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion and density centrifugation. pHEndECs are spindle shaped, contact inhibited, differentiate to form capillary-like tubes, are Ulex Europaeus Agglutinin 1 and von Willebrand Factor positive and endocytose acetylated low density lipoprotein. They also express alkaline phosphatase, γ-glutamyl transpeptidase, glucose transporter-1, p-glycoprotein and low levels of cellular adhesion molecules. Culturing pHEndECs on collagen coated transwell inserts was used to develop the IVBNB. High transendothelial electrical resistances (~160Ω.cm2; maximal 12 days after seeding) and low solute permeability to fluoresceinated high molecular weight (70 kDa) dextran (~0.7%) were seen. Electron microscopy demonstrated intercellular tight junctions and 50-100 nm-diameter pinocytic vesicles. These features are consistent with the BNB. This model provides an avenue to study mechanisms of leukocyte entry into peripheral nerves.

Mobilization of Lysosomal Calcium Regulates the Externalization of Phosphatidylserine during Apoptosis

A hallmark of apoptotic cells is the Ca2+-dependent appearance of phosphatidylserine (PS) at the cell surface as a result of its redistribution from the inner-to-outer plasma membrane leaflet. Although endoplasmic reticulum and mitochondrial Ca2+ are known to participate in apoptosis, their role in PS externalization has not been established. In this study, several organelle-specific fluorescent markers and Ca2+-sensitive probes were used to identify the source of Ca2+ critical to PS externalization. By employing Rhod-2AM, fluorescein-labeled high molecular weight dextran, and Calcium Green 1, we provide evidence that lysosomes respond to apoptotic stimuli by releasing their luminal Ca2+ to the cytosol. Cells treated with the cytosolic phospholipase A2 inhibitor, cPLA2alpha, had no effect on caspase activation but exhibited a significant decrease in lysosomal Ca2+ release and externalization of PS in response to apoptotic stimuli. Similarly, cells depleted of lysosomal Ca2+ underwent programmed cell death yet failed to externalize PS. These data indicate that although Ca2+ release from other intracellular organelles to the cytosol is adequate for apoptosis, the release of Ca2+ from lysosomes is critical for PS externalization.

Calcineurin inhibitor toxicity in a renal transplant recipient.

A 40-year-old Caucasian male who suffered end-stage renal disease due to unknown cause received a living-related renal allograft on 28 July 2008. Rabbit anti-thymocyte globulin was utilized for immediate post-transplant immunosuppressive induction, followed by maintenance immunosuppression with tacrolimus, mycophenolate mofetil and prednisone. Metoprolol was also administered for hypertension. Nadir serum creatinine occurred on post-transplant day number 7 (serum creatinine 2.2 mg/dL). At that time, the blood tacrolimus level was 14.8 ng/mL (reference range 5.0–20.0 ng/mL). The patient's serum creatinine abruptly rose to 2.6 mg/dL 3 days later, at which time his blood tacrolimus level was 46.8 ng/mL. A renal allograft biopsy (Figures ​(Figures11 and ​and2—see2—see Online Supplementary Material for colour illustrations) was performed a few days later, which revealed isometrically vacuolated proximal tubular epithelial cells and focal nodular arteriolar hyaline. The tubular epithelial cell isometric vacuolization seen was very focal, affecting only a few tubules in the biopsy sample. Isometric vacuolization describes the finding of numerous equally sized tiny clear cytoplasmic vacuoles. A diagnosis of acute calcineurin inhibitor toxicity (toxic tubulopathy) was made. These small, uniformly sized tubular epithelial cell cytoplasmic vacuoles are caused by toxic levels of the calcineurin inhibitors cyclosporine and tacrolimus and are due to dilatations of smooth endoplasmic reticulum by aqueous fluid [1,2,3]. This type of cytoplasmic hydropic change is light microscopically indistinguishable from that seen in osmotic nephrosis caused by hypertonic sucrose solutions, mannitol, high molecular weight dextrans, radiocontrast material or intravenous immunoglobulin. However, in contrast to those seen in calcineurin inhibitor toxicity, isometric vacuoles induced by the latter agents represent distended lysosomes rather than dilated smooth endoplasmic reticulum [4]. Ethylene glycol poisoning and hypokalaemia cause very large, coarse cytoplasmic vacuoles, easily distinguished by light microscopy from the numerous tiny vacuoles seen in calcineurin inhibitor toxicity and osmotic nephrosis [4]. In the case of calcineurin inhibitor toxicity, the typical cytoplasmic hydropic change may be so focal as to affect only a few tubules within a given biopsy [3]. Another, more serious acute toxic effect of calcineurin inhibitors is thrombotic microangiopathy, which was not seen in this biopsy. The patient's tacrolimus dose was lowered, followed by a reduction in his blood tacrolimus level and serum creatinine (2.1 mg/dL, 2 weeks post-biopsy). Fig. 1 Renal biopsy, stained with haematoxylin and eosin, reveals isometric vacuolization of proximal tubular epithelial cells. Tubular epithelial cell vacuolization is only focal (arrow), as seen by surrounding normal appearing tubules (original magnification ... Fig. 2 High-power view of involved renal tubule reveals marked epithelial cell swelling due to numerous tiny clear cytoplasmic vacuoles (original magnification ×600). For colour illustration, please see online supplementary materials.

Depletion of high molecular weight dextran from the red cell surface measured by particle electrophoresis

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biological and biophysical interest, yet the mechanistic details governing the process are still being explored. A depletion model has been proposed for aggregation by the neutral polyglucose dextran and its applicability at high molecular weights has been recently documented. In the present study the depletion of high molecular weight dextrans on the red cell surface was measured as a function of polymer molecular mass (40 kDa-28 MDa), ionic strength (5 and 15 mM NaCl) and polymer concentration (< or =0.9 g/dL). The experimental data clearly indicate an increasing depletion effect with increasing molecular weight: the effects of medium viscosity on RBC mobility were markedly overestimated by the Helmholtz-Smoluchowski relation, with the difference increasing with dextran molecular mass. These results agree with the concept of polymer depletion near the RBC surface and lend strong support to a "depletion model" mechanism for dextran-mediated RBC aggregation. Our findings provide important new insight into polymer-RBC interactions and suggest the usefulness of this model for fundamental studies of cell-cell affinity and for the development of new agents to stabilize or destabilize specific bio-fluids.

Multiple Specificity of Human Serum Dextran-Binding Immunoglobulin: α (1→6)- and β (1→3)-linked Glucose and α (1→3)-linked Galactose in Natural Glycoconjugates are Recognized

Dextran-binding immunoglobulin (DIg) was detected in circulating immune complexes in young healthy blood donors. High molecular weight dextran or non-dialysable polysaccharides of edible sugar added to serum markedly increased DIg-containing immune complexes. DIg from plasma was purified by an improved affinity chromatography using Sephadex G-200 to increase yield. Recognition by DIg of α (1→6) glucans in polystyrene-coated dextran was inhibited totally and its less avid recognition of β (1→3) glucans in yeast and barley partially by 50 mM 1-O-methyl-α-D-glucoside. All fractions of non-dialysable edible sugar separated by electrophoresis were recognized by DIg. While purified DIg contained nearly equal amounts of IgM and IgG, DIg in immune complexes isolated after addition of dextran to serum was 72% IgM. Recognition of bovine thyroglobulin, tumour laminin, and peanut-agglutinin-binding bovine heart glycoproteins, all containing terminal α (1→3)-linked galactose epitopes, by DIg was inhibitable by 1-O-methyl-α-D-glucoside. Enzymatic removal or modification of the respective terminal α (1→3)-linked galactose groups in these glycoproteins abolished their recognition by the antibody. Results suggest that DIg recognizes α (1→6) glucans, α (1→3) - linked galactose epitopes and less avidly β (1→3) glucans. Results also point to a new immunomodulatory effect of non-dialysable sugar and dietary β-glucans.

Viscoelastic adhesive mechanics of aldehyde-mediated soft tissue sealants

Soft tissue sealants generally sacrifice adhesive strength for biocompatibility, motivating the development of materials which interact with tissue to a predictable and controllable extent. Crosslinked hydrogels comprising aminated star polyethylene glycol and high molecular weight dextran aldehyde polymers (PEG:dextran) display aldehyde-mediated adhesion and readily tunable reactivity with soft tissue ex-vivo. Evaluation of PEG:dextran compositional variants revealed that the burst pressure of repaired intestinal wounds and the extent of material-induced tissue deformation both increase nonlinearly with formulation aldehyde content and are consistently within the desired range established by traditional sealants. Adhesive test elements featuring PEG:dextran and intestinal tissue exhibited considerable viscoelasticity, prompting use of a standard linear solid (SLS) model to describe adhesive mechanics. Model elements were accurately represented as continuous functions of PEG:dextran chemistry, facilitating prediction of adhesive mechanics across the examined range of compositional formulations. SLS models of traditional sealants were also constructed to allow general correlative analyses between viscoelastic adhesive mechanics and metrics of sealant performance. Linear correlation of equilibrium SLS stiffness to sealant-induced tissue deformation indicates that dense adhesive crosslinking restricts tissue expansion, while correlation of instantaneous SLS stiffness to burst pressure suggests that the adhesive stress relaxation capacity of PEG:dextran enhances their overall performance relative to traditional sealants.

Partitioning of charged and neutral dextran-dye derivatives in biphasic cellulose nanocrystal suspensions

The partitioning behaviour of dye-labeled dextrans of high molecular weight in aqueous suspensions of native cellulose nanocrystals was studied. Cellulose concentrations lie in the isotropic–nematic coexistence region. Blue dextrans of various molecular weights and degrees of substitution of dye molecules (anionic Cibacron blue 3G-A) were investigated. Increasing the total concentration of blue dextran and degree of dye substitution led to increasing partition coefficients. Increasing dextran molecular weight resulted in higher partition coefficients, in agreement with theory. Partition coefficients were larger than predicted theoretically using a second virial coefficient approximation. Electrostatic and entropic contributions to the partition coefficient of blue dextran are discussed. Dextrans labeled with neutral fluorescein isothiocyanate did not partition preferentially in this system.Key words: partition coefficient, cellulose nanocrystals, dextrans, degree of substitution, polyelectrolyte.

Effects of Salidroside on Hemorheology and Myocardial Ischemic Reperfusion Injury in Isolated Heart of Rats

ABSTRACT AIM To study the effects of salidroside on hemorheology and myocardial ischemic reperfusion injury in isolated heart of rats. METHODS High-molecular weight dextran was used to develop the model of acute blood stasis in rats, and the effects on hemorheology were observed. The effects of salidroside on the coronary blood flow and cardiac contractility of isolated rat heart were observed using ischemia/reperfusion model. RESULTS Salidroside (6, 12, 24 mg·kg −1 ) can effectively prevent the increases of the whole blood viscosity (high, middle and low shear rates), plasma viscosity, hematocrit and fibrinogen; Salidroside(1.6, 2.0, 2.5 mg·L −1 ) can increase the coronary blood flow and enhance the myocardial contractility of the isolated rat heart significantly. CONCLUSION Salidroside can improve hemorheology of rats in the acute blood stasis model, and it has protective effects on myocardial ischemic reperfusion injury in isolated rat heart.

Intravital Microscopic Analysis of Vascular Perfusion and Macromolecule Extravasation after Photodynamic Vascular Targeting Therapy

Photodynamic therapy (PDT), involving the combination of a photosensitizer and light, is being evaluated as a vascular disrupting therapy and drug delivery enhancement modality based on its effects on vascular perfusion and barrier function. Since tumor vasculature is the common route for the delivery of both blood and therapeutic agents, it is important to compare the effects of PDT on blood perfusion and substance transport. Tumor blood cell velocity and the extravasation of high molecular weight dextran molecules were continuously monitored by intravital fluorescence microscopy for up to 60 min after PDT using three doses of verteporfin in the MatLyLu prostate tumor model. PDT induced tumor perfusion disruption via thrombus formation. PDT using a higher dose of verteporfin was more effective in inhibiting blood perfusion while a lower dose verteporfin-PDT was more potent in enhancing dextran extravasation. The increase in dextran extravasation induced by PDT was dependent upon dextran molecular weight. A lower molecular weight dextran obtained a higher tumor accumulation after PDT than a higher molecular weight dextran. PDT with verteporfin had different effects on tumor vascular perfusion versus the extravasation of macromolecules. Optimal PDT conditions should be adjusted based on the therapeutic application.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Effect of Dextran 500 on radial migration of erythrocytes in postcapillary venules at low flow rates

Recently, we reported that collision efficiency (fraction of total collisions that result in the formation of aggregates) between red blood cells was an important factor in the formation of aggregates in postcapillary venules. In the present study, we focus on how high molecular weight dextran influences the overall radial migration trend of red blood cells in the postcapillary venule along a longitudinal distance of 50 microm from the bifurcation which would in turn affect collision behavior of these cells. A radial migration index, which defines the extent of radial migration of individual cells relative to the vessel center, was found to have a larger magnitude after infusion of dextran (1.9 +/- 2.73) compared to that before dextran infusion (1.48 +/- 3.89). This implied that dextran-induced aggregation might provide an external force to actively move cells towards the centerline of the vessel, which could contribute to the greater number of red blood cells participating in collision (16% increase) and aggregate formation. Further analysis of the collision behavior of individual red blood cells revealed that collision frequencies of individual cells decreased from a wide range (1 to 14) to a narrow range (1 to 5) after dextran treatment, indicating the alteration of collision behavior of red blood cells by the presence of aggregates along the flow stream.

Synthesis of (Dex-HMDI)-g-PEIs as effective and low cytotoxic nonviral gene vectors

Gene vectors, (dextran-hexamethylenediisocyanate)-g-polyethylenimines ((Dex-HMDI)-g-PEIs), were synthesized through grafting low molecular weight (800 Da) branched polyethylenimine (PEI) to HMDI functionalized dextrans with two different molecular weights. The buffer capabilities of (Dex-HMDI)-g-PEIs were examined by acid–base titration. The titration profiles show that both (Dex-HMDI)-g-PEIs have the similar buffer capability regardless of the different molecular weight of dextran. Physiochemical characteristics of (Dex-HMDI)-g-PEI/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and ζ-potential measurements. The result of gel electrophoresis suggests that both (Dex-HMDI)-g-PEIs are able to condense DNA efficiently at N/P ratios higher than 4. The particle sizes of (Dex-HMDI)-g-PEI/DNA complexes are around 160–250 nm, and the surface charges are around 19–23 mV at the N/P ratios ranging from 10 to 60. The morphology of complexes was observed by scanning electron microscopy (SEM) and the images show that nano-sized complexes display a regular spherical shape. In vitro cell viability and transfection were evaluated in 293T and HeLa cells using 25 kDa PEI as a control. The cytotoxicity of (Dex-HMDI)-g-PEIs is lower than that of 25 kDa PEI. The gene transfection efficiency of (Dex-HMDI)-g-PEI/DNA complexes at certain N/P ratios in 293T cells is higher than or comparable to 25 kDa PEI/DNA complex at its optimal N/P ratio of 10. In addition, comparing with (Dex-HMDI)-g-PEI with a high molecular weight dextran, (Dex-HMDI)-g-PEI with a low molecular weight dextran demonstrates lower cytotoxicity and higher transfection efficiency.

Red cell aggregation as a factor influencing margination and adhesion of leukocytes and platelets

Leukocytes and platelets must adhere to the wall of blood vessels to carry out their protective functions. Rheological factors influencing these processes are the delivery of the cells to the wall, referred to as margination, and the local shear rates and stresses at the wall. Margination requires leukocytes and platelets to be excluded from the central flow of the much more numerous red blood cells. This exclusion may be influenced by red cell aggregation. Red cell aggregation also influences development of plug flow in small vessels, which in turn modifies the wall shear rate and stress from those expected in ideal Poiseuille flow. Promotion of aggregation by added agents such as high molecular weight dextrans or by reduction in shear rate, increases margination of leukocytes and efficiency of attachment to the vessel wall. Interestingly, however, fewer studies exist for platelets, and these suggest that margination is actually promoted by increasing shear rate. Direct studies of the effects of red cell aggregation on platelets are required, but it appears that aggregation has different effects on delivery of platelets compared to leukocytes. These differences may represent adaptations for efficient adhesion of leukocytes and platelets in different regions of the circulation.