Dextran sulfate nanoparticles as a theranostic nanomedicine for rheumatoid arthritis.

With the aim of developing nanoparticles for targeted delivery of methotrexate (MTX) to inflamed joints in rheumatoid arthritis (RA), an amphiphilic polysaccharide was synthesized by conjugating 5ß-cholanic acid to a dextran sulfate (DS) backbone. Due to its amphiphilic nature, the DS derivative self-assembled into spherical nanoparticles (220 nm in diameter) in aqueous conditions. The MTX was effectively loaded into the DS nanoparticles (loading efficiency: 73.0%) by a simple dialysis method. Interestingly, the DS nanoparticles were selectively taken up by activated macrophages, which are responsible for inflammation and joint destruction, via scavenger receptor class A-mediated endocytosis. When systemically administrated into mice with experimental collagen-induced arthritis (CIA), the DS nanoparticles effectively accumulated in inflamed joints (12-fold more than wild type mice (WT)), implying their high targetability to RA tissues. Moreover, the MTX-loaded DS nanoparticles exhibited significantly improved therapeutic efficacy against CIA in mice compared to free MTX alone. Overall, the data presented here indicate that DS nanoparticles are potentially useful nanomedicines for RA imaging and therapy.

Determination of residual dextran sulfate in protein products by SEC-HPLC.

Dextran sulfate is a polyanionic derivative of dextran, produced by esterification of dextran with chlorosulphonic acid. Dextran sulfate with an average molecular weight of 8000Da can be added to the cell culture to inhibit binding of proteins to cells, increasing cellular growth and productivity. Residual dextran sulfate levels must be monitored during the purification process development to insure clearance. A size-exclusion chromatography based HPLC assay has been developed for the separation and quantitation of dextran sulfate in a highly concentrated purified protein drug substance sample. Trichloroacetic acid (TCA) was used to precipitate the protein and separate the dextran sulfate. Detection and quantitation of dextran sulfate was achieved by post column reaction with dimethylene blue to form a metachromatic complex that absorbs visible light at 530nm. The quantitation limit (LOQ) was determined to be 1.5µg/mL dextran sulfate in high concentration protein samples.

The development of an FIA-CD strategy for screening sulfated polysaccharides using antimalarial drugs and related species as probes.

Heterogeneous sulfated polysaccharides have attracted significant attention in light of their various biological activities. However, recent events involving heparin have dramatically illustrated that several analytical challenges exist in accounting for such species. In this case, tainted heparin was associated with acute reactions that lead to numerous deaths. Researchers were forced to use time-consuming, sophisticated techniques (e.g., enzymatic digestion, NMR, CE, HPLC, MS, etc.) to identify the cause of these adverse effects. Extensive investigations ultimately showed oversulfated chondroitin sulfate, a semi-synthetic sulfated polysaccharide, to be present in the contaminated samples. These events highlighted the need for a new generation of screening techniques. In this work, we report the development of a screening strategy that exploits unique circular dichroism features observed as a function of association between investigated polymers and judiciously selected probe molecules (i.e., chloroquine, N1-(7-chloro-4-quinolinyl)-N3-methyl-1,3-propanediamine, quinacrine, and N2-9-acridinyl-N1,N1-dimethyl-1,2-ethanediamine). Application of obtained spectropolarimetry results to a flow injection analysis circular dichroism platform allowed for the establishment of linear polysaccharide response curves for dextran sulfate, heparin, and oversulfated chondroitin sulfate in the low micromolar range. Lastly, through additional work with heparin, the proposed method was shown to be capable of rapidly screening sulfated polysaccharide samples for closely related impurities.

[Distribution of biological particles in two-phase systems of water soluble polymers and salts]. / Raspedelenie biologicheskikh chastits v dvukhfaznykh sistemakh vodorastvorimykh polimerov i solei.

The paper discusses the problems of purification of Provachek's rickettsias used to prepare typhus vaccines and diagnostic kits. The currently available agents contain many admixtures of rickettsias products, more commonly yolk and chick embryo yolk sack cell detritus, which make it impossible to determine the true antigenic properties of rickettsias in the context of protection and diagnosis. By applying the well-known principle of distribution of biological particles in the two-phase systems of water soluble polymers, investigations were made to examine the distribution of rickettsias and their infected biological mass in the system: polyethylene glycol-6600, sodium-500 dextran sulfate, potassium phosphate, sodium chloride in order to prepare agents of pure rickettsias. It has been found that rickettsias are distributed in the polymer-enriched phase, their number and the degree of purity when isolated from the infected biological mass depends on the capacity of an interphase wherein tissue detritus sorption occurs.

Detection of polyanion-restricted anti-histone antibodies in patients with systemic lupus erythematosus.

The nature of the antibodies responsible for lupus erythematosus (LE) cells in systemic lupus erythematosus (SLE) remains obscure. We examined whether polyanion-restricted anti-histone antibodies were present in serum of patients with SLE using Western blotting analysis. Dextran sulfate or alginate was used as a polyanion compound in place of DNA. Antibodies which recognized dextran sulfate-histone complexes were present in serum of patients with SLE (17/34, 50%). These antibodies were detected in most SLE patients positive for LE cells (17/18, 94%) but not in those negative for LE cells or in patients with other collagen diseases. Similar results were obtained using alginate-histone complexes as antigens for Western blotting analysis. The antibodies to dextran sulfate-histone or alginate-histone complexes in serum of SLE patients were completely absorbed by treatment of serum with DNA-histone complexes, while they were unaffected by treatment with DNA only. The presence of antibodies to free histones and dextran sulfate-histone complexes did not seem to be related to the titer of anti-single stranded DNA antibody and anti-double stranded DNA antibody. We demonstrated the presence of polyanion-restricted antibodies in SLE, which may be responsible for the LE factor.

Uptake of dextran sulphate by glomerular intracellular vesicles during kidney ultrafiltration.

Dextran sulphate is often used as a model for albumin in understanding capillary-tissue exchange and the charge selective nature of the capillary wall, particularly in kidney ultrafiltration. In investigating the mechanism of transport in the kidney, autoradiographic analysis of the distribution of iodinated dextran sulphate in perfused rat kidneys demonstrates the preferential accumulation of the probe in the glomerular capillary wall. Tritium-labeled dextran sulphate is found to be specifically taken up by intracellular 20 to 40 nm vesicles that can be isolated post-perfusion. The molecular weight profile of vesicular dextran sulphate demonstrates that the dextran sulphate containing vesicles are from vascular glomerular cells. Dextran is not taken up by the vesicles. These results suggest that the apparent charge selectivity associated with the transglomerular transport of the dextran sulphate is associated, in part, with cell-mediated processes at the glomerular level.

A novel method for differentiating dextran sulfate from related sulfated polysaccharides.

A method is described for unequivocal identification of dextran sulfate, based on combined chemical desulfation and dextranase enzymolysis of dextran sulfate moieties to isomaltose, a specific indicator of dextran-type precursors. The method was developed using high-resolution (300 MHz) 1H NMR spectroscopy for assurance of the molecular transformations, identification, and estimation of the hydrolysis products. Overall conversion of approximately 80% of highly sulfated and moderately sulfated dextran sulfates was realized. Both 2-D 1H and 13C NMR spectra of a dextran sulfate (MW 500,000) clarified the extent of sulfation (75%) at C-4 and confirmed that sulfation at positions C-2 and C-3 was virtually complete. Estimation of the hydrolysis products (isomaltose, major; alpha-D-glucose, minor) is not restricted to 1H NMR now that the desulfation-enzymolysis methodology has been established; rather, it can be performed using HPLC or GLC (with derivatization).