Immobilization of heparin on polysulfone surface for selective adsorption of low-density lipoprotein (LDL)

A versatile method was developed to immobilize heparin covalently on polysulfone sheets (PSu) to achieve selective adsorption of low-density lipoprotein (LDL). This was achieved by activation of PSu with successive treatments of chlorodimethyl ether and ethylenediamine, and subsequent chemical binding of heparin with bifunctional linker molecules. A heparin density up to 0.86 μg cm^?2 on a dense PSu film was achieved. The modified PSu films were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The hydrophilicity of the PSu film was improved greatly by covalent immobilization of heparin. The water contact angle of PSu film was decreased from 86.6 ± 3.7° to 50.5 ± 3.2° after binding of 0.36 μg cm^?2 heparin. An enzyme-linked immunosorbent assay was used to measure the binding of LDL on plain and modified PSu films. It was found that the heparin-modified PSu film could selectively recognize LDL from binary protein solutions. Furthermore, it was possible to desorb LDL from heparinized PSu, but not from plain PSu, with heparin, sodium chloride or urea solution, which indicates a selective but reversible binding of LDL to heparin. The results suggest that heparin-modified PSu membranes are promising for application in simultaneous hemodialysis and LDL apheresis therapy.     

Surface glycosylation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) membrane for selective adsorption of low-density lipoprotein

A novel method of constructing a glycosylated surface on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] membrane surface for the selective adsorption of low-density lipoprotein (LDL) was developed, which involved the photoinduced graft polymerization of acrylic acid followed by the chemical binding of carboxyl groups with glucosamine in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride and N-hydroxy-succinimide. The chemical structures of the fabricated membranes were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Zeta potential and water contact angle measurements were performed to investigate the surface charge and wettability of the membranes, respectively. An enzyme linked immunosorbent assay was used to measure the LDL adsorption on the plain and modified membrane surfaces. It was found that the surface glycosylation of P(3HB-co-4HB) membrane greatly enhanced the affinity interactions with LDL and the absorbed LDL could be easily desorbed with eluents, indicating a specific and reversible binding of LDL to the surface. Furthermore, the hemocompatibility of glycosylated membrane was improved as examined by platelet adhesion. The results suggest that the glycosylated P(3HB-co-4HB) membrane is promising for application in LDL apheresis therapy.     

Diffusion characteristics of alginate membranes

The diffusive properties of alginate membranes prepared by the interfacial reaction of sodium alginate solution with gypsum substrates were studied. The penetrants of primary importance for the above study were methyl methacrylate (MMA) and water, since alginate membranes are used as mould release agents, during the casting of poly(methyl methacrylate) (PMMA) in gypsum moulds in a water bath. Vapour sorption measurements were carried out at various temperatures. The effect of temperature and the combined action of water and MMA on the swelling behaviour of the membrane was also investigated. The results indicate that the water sorption of alginate membranes is non-Fickian, in spite of the linearity of the curves obtained by plotting Mt/M infinity versus t 1/2, at least for a short time period. The membrane treatment with methyl methacrylate, leads to considerably lower equilibrium sorption, possibly because of the formation of a thin film of poly(methyl methacrylate) on the membrane surface.     

Equilibrium adsorption of LDL and gold immunoconjugates to affinity membranes containing PEG spacers

The objective of this study was to provide insight into the effects of spacer chemistry on immunoaffinity separations for the capture of large macromolecules and biological complexes. Immunoaffinity membranes were prepared by immobilization of immunoglobulin G (IgG) to flat sheet microporous membranes. Two different systems were examined: immobilized IgG for the immunoadsorption of human low-density lipoprotein (LDL) and immobilized IgG for the immunoadsorption of gold particle immunoconjugate. The IgG was immobilized either directly to the membrane or via a polyethylene glycol (PEG) spacer. Adsorption of LDL was significantly greater for anti-LDL IgG immobilized via PEG than for IgG immobilized directly to the membrane. With the PEG spacer, the adsorption capacity for LDL matched the theoretical density of a monolayer of LDL particles on the membrane surface. The gold particle immunoconjugate, similar in size to LDL, was examined as a generalized model of restrictions to immunoaffinity adsorption of large (>20 nm) biological complexes. Adsorption of gold particles was greater for IgG immobilized via PEG than for IgG immobilized directly to the membrane. It is postulated that the PEG spacer allows lateral movement of the immobilized IgG and dense monolayer packing of adsorbed particles on the membrane surface. These results are pertinent to the removal of LDL from human plasma and the purification of gene therapy delivery vectors, viral vaccines, and other large biological complexes.     

A crossover study of the acrylonitrile-co-methallyl sulfonate and polysulfone membranes for elderly hemodialysis patients: the effect on hemodynamic, nutritional, and inflammatory conditions

Many maintenance hemodialysis (MHD) patients have recently been treated with high flux (HF) dialysis membranes such as polysulfone (PSu) membranes. However, the appropriateness of HF for elderly MHD remains to be elucidated. In order to estimate hemodialysis (HD) efficiency, the hemodynamic condition during HD, and the nutritional status, 28 elderly MHD patients were treated with PSu for 3 months. After this, the patients were switched to acrylonitrile-co-methallyl sulfonate (AN69) membranes for the next 3 months and then returned to PSu for another 3 months. Reduction ratio of inflammatory cytokines (interleukin [IL]-6) by AN69 was significantly higher than the reduction ratio by PSu. After 3 months with AN69, the serum total protein, albumin, and cholesterol levels significantly increased, and after switching back to PSu, the levels returned to baseline. Furthermore, the frequency of saline used to treat episodes of hypotension during HD significantly decreased in the AN69 period. In elderly MHD patients, it was possible to achieve improvements in both malnutrition and chronic inflammatory conditions with AN69. This suggests that AN69 may be the preferred membrane for elderly MHD, because it stabilizes the hemodynamic condition and demonstrates a higher removal of inflammatory cytokines during HD.     

Chitosan-coated alginate membranes for cultivation of limbal epithelial cells to use in the restoration of damaged corneal surfaces

Some chemicals or thermal burns may result in abnormal reepithelialization by conjunctival epithelial cells and it causes different types of damage on the cornea surface. When reepithelialization does not occur, chronic inflammation and neovascularization develop, often leading to stroma scarring and/or ulceration. The aim of this study is to restore the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of the limbal epithelial cells over the different compositions of composite membranes. The composite membranes were prepared by coating the alginate membrane with chitosan. In this method, alginate membrane was prepared by precipitation of the sodium alginate solution in calcium chloride solution. Alginate membranes were washed, dried and immersed into the chitosan solutions to prepare composite membranes. The composite membranes were characterized based on their morphology, hydrophilicity, swellability, and chemical structure. In the last part of the study, composite membranes were used as base matrices for limbal epithelial cell cultivation. The cell cultivation on polymeric membranes was investigated as the in vitro studies. In these studies cell attachment, spreading and growth on polymeric membranes were evaluated.     

Adsorption of human immunoglobulin to implantable alginate-poly-L-lysine microcapsules: effect of microcapsule composition

Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the immune response towards the implants. Here, we investigate the adsorption of immunoglobulins (IgG, IgM, and IgA) onto the surface of APA microcapsules in vitro after their exposure to human serum and peritoneal fluid. Immunoglobulins (Ig) are considered to be opsonizing proteins, thus they tend to mediate inflammation when adsorbed to foreign surfaces. Ig adsorption was monitored using direct immunofluorescence. The amount of Ig adsorbed to the microcapsule surface was not significantly influenced by the guluronic acid content nor the purity level of the alginate, although microcapsules of intermediate-G purified alginate corresponded with the lowest adsorption levels. Ig adsorption was negligible when the poly-L-lysine membrane was omitted, suggesting that positive charges at the microcapsule surface are responsible for binding Ig.     

Usefulness as guided bone regeneration membrane of the alginate membrane

Alginate membrane is a new bioabsorbable, guided bone regeneration (GBR) membrane, which is placed directly on the surface of the bone defect. It is designed to drop a calcium chloride aqueous solution into the bone defect, which is filled with sodium alginate aqueous solution. Alginate membrane is an excellent agent for this procedure due to its close assimilation to the surface of the bone. In this study, we evaluated the short-term biocompatibility of alginate membrane in the bone defects of rat tibiae. GBR membrane availability was also examined. Consequently, we found that the healing process in bone defects covered with an alginate membrane was delayed in comparison with that of controls, however, the defect was restored to nearly original condition. In contrast, in the controls, bone defect repairs exhibited partitioning as a result of connective tissue involvement. Furthermore, we observed a relation between the sodium alginate concentration and the rate of absorption of the sodium alginate membrane. Absorption of a 1.5% sodium alginate membrane was slow. As a result, the compound was not absorbed completely and bone repairs resembled an hourglass. Moreover, the inflammatory response was absent surrounding the alginate membrane. The present findings suggested that the alginate membrane functions effectively as a GBR membrane. In addition, the alginate membrane derived from 3% calcium chloride and 1% sodium alginate was most suitable as a GBR membrane.     

Poly(ethylene oxide)-graft-poly(L-lysine) copolymers to enhance the biocompatibility of poly(L-lysine)-alginate microcapsule membranes.

A graft copolymer having poly(L-lysine) (PLL) as the backbone and monomethoxy poly(ethylene glycol) (MPEG) as pendent chains was synthesized. This polycationic copolymer was used to form microcapsules with sodium alginate, a polyanion. Microcapsules and model surfaces formed with PLL-graft-MPEG demonstrated reduced protein adsorption, complement binding and cell adhesion in vitro compared to materials with unmodified PLL. Microcapsules with PLL-g-MPEG on the surface were seen to be much more biocompatible than the widely used alginate/PLL/alginate microcapsule in a mouse intraperitoneal implant model. The graft copolymers demonstrated a lower affinity for alginate and increased microcapsule permeability more than PLL. To correct this, pentalayered alginate/PLL/alginate/PLL-g-MPEG/alginate microcapsules were fabricated, and these demonstrated both appropriate permselectivity and enhanced biocompatibility.     

Self-setting barrier membrane for guided tissue regeneration method: initial evaluation of alginate membrane made with sodium alginate and calcium chloride aqueous solutions.

Alginate membrane was proposed as a self-setting barrier membrane that can be used for guided tissue regeneration (GTR). The alginate membrane can be prepared and placed at the bone defect during the surgical procedure. The procedure consists of two simple steps. First, the bone defect is filled with sodium alginate (Na-Alg) aqueous solution. Then calcium chloride aqueous solution is dropped on the surface of the Na-Alg aqueous solution. An alginate membrane is formed on the bone defect, keeping the inside of the bone defect filled with unreacted Na-Alg aqueous solution. In this investigation, a preliminary animal study was conducted for an initial evaluation as to whether or not the alginate membrane can be used as a barrier membrane for the GTR method. Bone defects were made in the tibiae of 15-week-old rats. The alginate membrane was made on the surface of existing bone by filling the defect with Na-Alg aqueous solution and then dropping calcium chloride aqueous solution onto the surface of the Na-Alg solution. Four weeks after surgery, the bone defect was found to be reconstructed with new bone when the defect had been covered with alginate membrane whereas the bone defect was filled only with connective tissue when it had been kept open. We concluded, therefore, that this alginate membrane may be a useful barrier membrane when the GTR method is employed.     

Protein binding properties of surface-modified porous polyethylene membranes

In this study, we quantified the adsorption of immunoglobulin G (IgG) protein onto several polyelectrolyte-modified sintered porous polyethylene (PPE) membranes. The polymer surfaces had both cationic and anionic charges obtained via the adsorption of polyethylenimine (PEI) and polyacrylic acid (PAA), respectively, onto plasma-activated PPE. The amount of IgG adsorption was determined by measuring the gamma radiation emitted by [125I]-IgG radio labeled protein. By studying the impact of pH and ionic strength on IgG adsorption, we attempted to characterize the role and nature of the electrostatic interactions involved in the adsorption process to better understand how these interactions were influenced by the charge and structure of immobilized polyelectrolyte complexes at modified membrane surfaces. We were able to show that surface modification of PPE membranes with adsorbed PEI monolayers and PEI-PAA bilayers can greatly improve the IgG binding ability of the membrane under optimized conditions. We also showed that the observed improvement in the IgG binding is derived from electrostatic interactions between IgG and the polyelectrolyte surface. In addition, we found that the greatest IgG adsorption occurred when the IgG and the surface possessed predominantly opposite charges, rather than when the surface possessed the greatest electrostatic charge. Finally, we have found that the molecular weight of the terminating polyelectrolyte has a noticeable effect upon the electrostatic interactions between IgG and the PEI-PAA bilayer-modified PPE surfaces.     

Human lipoprotein binding to schistosomula of schistosoma mansoni. Displacement by polyanions, parasite antigen masking, and persistence in young larvae.

It was previously shown by the authors that the binding of human low-density lipoprotein (LDL) to the surface of schistosomula inhibits the binding of human anti-schistosomal antibodies and is inhibited by suramin. Here, three questions were considered. 1) Are LDLs bound to schistosomula displaced from the membrane by polyanions? 2) Does bound LDL mask or hide antigens recognized by human anti-schistosomal antibodies? 3) Is LDL, binding capability present when the larvae enter the blood stream? The first question was tested by measuring the percentage of the schistosomular surface membrane covered by LDL after exposure to LDL with or without dextran sulfate or suramin. The bound LDL was visualized with polyclonal goat anti-human apolipoprotein B (anti-apo B) antibodies and peroxidase-conjugated secondary antibodies. After overnight culture in 20 micrograms/300 microliters LDL, 84.0% +/- 0.3% of the parasite surface was covered by LDL reaction product. When the polyanions suramin or dextran sulfate were added to the cultures for 30 minutes, only 59.7% +/- 4.9% of the surface was covered by reaction product, demonstrating that the LDL was partially displaced from the membrane by these compounds. The second question was tested by measuring the binding of human and mouse monoclonal anti-schistosomal antibodies before and after exposure to LDL, with or without partial removal of the bound LDL by suramin. LDL partially inhibited antibody binding in a reversible fashion. The LDL clearly masked parasite antigens, most probably by steric hindrance. However, there may be competitive inhibition of antibody binding by the LDL as well, because human anti-schistosomal antibodies inhibited LDL binding to worms and both human anti-schistosomal antibody and LDL binding to schistosomula were inhibited by suramin. Finally, the third question was tested by quantitative immunofluorescence. The LDL binding capability persisted and nearly doubled by 72 hours after transformation from cercariae. These experiments demonstrated that LDL bound to the surface of schistosomula through the time they enter the blood stream. LDL bound to the parasite surface may help the parasite to evade antibody-dependent cytotoxic reactions by masking parasite antigens.     

Surface engineering of poly(DL-lactic acid) by entrapment of alginate-amino acid derivatives for promotion of chondrogenesis

Alginate-amino acid derivatives were explored to engineer poly(DL-lactic acid)(PDL-LA) as glycocalyx-like surface to promote cell adhesion and growth. Four different kinds of alginate-amino acid derivatives were synthesized to mimic the glycocalyx of cell membrane to promote chondrogenesis. The alginate-amino acid derivatives were characterized by FT-IR, 1H NMR and UV spectra and the amino acid content on alginate-amino acid derivatives was given by ninhydrin-UV method. A new strategy, entrapment, was then employed to modify PDL-LA membranes with alginate and its amino acid derivatives. The results of XPS, ATR-FTIR and contact angle confirmed that a stable thin film of alginate and its amino acid derivatives can be entrapped on the surface of PDL-LA membrane. The chondrocyte cytocompatibility test and MTT assays indicated that the alginate-amino acid derivatives modified PDL-LA membranes could promote chondrogenesis. The novel surface treatment method may have potentials for tissue engineering and other biomedical applications.     

Chitosan adsorbents carrying amino acids for selective removal of low density lipoprotein

Chitosan beads carrying various amino acids (a total of 12 kinds) were synthesized through quite simple procedures for selective removal of low density lipoprotein (LDL). Macroporous chitosan beads were prepared by the phase-inversion method, to which the amino acids were then coupled respectively, via either ethyleneglycol diglycidylether (EGDE) or epichlorohydrin (ECH). Among the amino acids used, in vitro tests proved L-Trp to be the best ligand for binding LDL. The adsorbent, which was prepared by coupling L-Trp to the chitosan beads via EGDE, demonstrated satisfactory adsorption performance for selective removal of LDL in human plasma.     

The effects of detergent on the enzyme-linked immunosorbent assay (ELISA) of blood group substances

The detergents 1-0-n-octyl-beta-D-glucopyranoside (OBG) and sodium n-dodecyl sulphate (SDS) have been used to extract blood group substances from human erythrocyte membranes for detection by enzyme-linked immunosorbent assay (ELISA). The effect of detergent concentration on the extraction process and detection by ELISA have been investigated. Detergent extraction increased the ELISA response relative to response from membrane suspensions approximately 1000-fold. Optimum responses occurred using detergent concentrations near the critical micelle concentration (cmc) for OBG and below the cmc for SDS. High detergent concentrations interfered with the ELISA but this effect was reduced by dilution of the extracts before adsorption of antigen on the microtitre wells. The interference effects of detergent on ELISA were also investigated using ovarian cyst glycoproteins as antigen. It was found that detergents inhibit the assay at the initial stage by competing with antigens for adsorption sites on the microtitre well surface and that subsequent detergent can displace pre-bound antigen. The results are discussed in terms of detergent binding to proteins (and glycoproteins) in relation to free (unbound) detergent concentration.     

Testing protein permeability of dialysis membranes using SDS-PAGE

BACKGROUND: Permeability of dialysis membranes for high molecular weight compounds should be similar to that of the glomerular membrane in order to remove uremic toxins like the human kidney does. In order to evaluate permeability of high-flux dialysis membranes SDS-PAGE is applied for examination of filtrate of dialysers during routine dialysis with different membranes. METHOD: SDS-PAGE analysis is performed with silver staining method according to the modification of Melzer (5) and consecutive laser densitometry. RESULTS: The protein pattern of filtrate from dialysis membranes is similar to that of the glomerular membrane containing IgG, transferrin, albumin, alpha-1-microglobulin, retinol binding protein and beta-2-microglobulin. Comparing different membranes there are considerable differences depending on cut-off, charge and adsorption capacity of the particular membrane. In all membranes tested permeability of proteins decreases during one treatment session. CONCLUSION: Protein permeability of high-flux dialysis membranes is similar to the gloemerular membrane but modified according to pore-size, surface charge, adsorption and time on dialysis. In contrast to the glomerular membrane in each of the investigated membranes protein permeability decreases during function.     

Specific binding of human low-density lipoprotein to the surface of schistosomula of Schistosoma mansoni and ingestion by the parasite.

Low-density lipoproteins (LDL) may be important in human schistosomiasis because LDL bound to the surface of the parasite inhibits the binding of anti-schistosomal antibodies. Low-density lipoproteins also may serve as a source of lipids for the parasite membrane synthesis. Here LDL fluorescently labeled with carbocyanine dye (DiI-LDL) was used to measure the specificity of binding of LDL to the surface of schistosomula of Schistosoma mansoni and to examine the distribution of the LDL particles over time. DiI-LDL binding was saturable and specific, with strong inhibition by unlabeled LDL and apoB but not by apoA1, bovine serum albumin, or IgG, and only weak inhibition by high-density lipoproteins. Half of the bound DiI-LDL was displaced by unlabeled LDL. DiI-LDL remained bound on the surface of schistosomula for up to 36 hours in culture. However parasites also ingested both DiI-LDL and a second fluorescent LDL, Bodipy-LDL. Over time, both fluorophores appeared throughout the worm tissues, suggesting the LDL particles were breaking down and that the fluorophores and lipids originally contained within the LDL particle were partitioning throughout the worm. Thus human LDL appears to bind to the surface of schistosomula specifically. Ingested LDL appears to be broken down and may serve as a source of host lipids for the parasite.     

Tethering poly(ethylene glycol)s to improve the surface biocompatibility of poly(acrylonitrile-co-maleic acid) asymmetric membranes

To improve the surface biocompatibility, asymmetric membranes fabricated from poly(acrylonitrile-co-maleic acid)s (PANCMAs) synthesized by water-phase precipitation copolymerization were tethered (or immobilized) with poly(ethylene glycol)s (PEGs) by esterification reaction. Chemical changes on the membrane surface were characterized by Fourier transform infrared spectroscopy and elemental analysis to confirm the immobilization of PEG onto the PANCMA membranes. The hydrophilicity and blood compatibility of the PEG-tethered PANCMA membrane were investigated by water contact angle, water absorption, protein adsorption, plasma platelets adhesion and cell adhesion measurements, and the results were compared with the corresponding PANCMA membranes. It was found that, after the tethering of PEG, the hydrophilicity of the membrane can be improved significantly, and the protein adsorption, platelets adhesion and macrophage attachment on the membrane surface are obviously suppressed. Furthermore, not only the content of maleic acid in PANCMA, which influences the tethering density of PEG, but also the molecular weight of PEG has great effect on the surface modification of PANCMA membranes for biocompatibility.     

Blood compatible aspects of DNA-modified polysulfone membrane-protein adsorption and platelet adhesion

DNA was used as a biomaterial to modify the polysulfone (PSf) membrane by blending it with PSf. The blood compatibility of the membranes was then investigated. The water contact angle decreased, and the hydrophilicity increased when a single strand DNA was blended with PSf. Because of the hydrophilic surface, the DNA-blended PSf membranes had a lower protein adsorption than the PSf membrane, but it was not significantly decreased due to the interaction between the DNA and proteins. Circular dichroism (CD) spectroscopy was used to examine the changes in the secondary structure of the proteins after adsorption onto the polymer surface and desorption from the polymer surface into the SDS solution. The conformation of the proteins adsorbed onto the PSf membrane and desorbed from the PSf membrane significantly changed, but that of the proteins for the DNA-blended PSf membranes differed only slightly from the native one. The number of platelets that adhered on the surface of the DNA-blended PSf membranes was reduced compared to that on the PSf membrane. This suggested that DNA can be regarded as a biopolymer to modify PSf, and contributes to the hydrophilic and hemocompatible wipers on the surface of the hydrophobic PSf membranes.     

Biocompatible microcapsules with enhanced mechanical strength.

A block copolymer, (short-chain alginate)-co-MPEG, was synthesized and used for coating the capsular membranes of the photosensitive microcapsules. The resulted microcapsules exhibited an excellent mechanical strength. The permeability test results revealed that the capsular membrane was freely permeable to cytochrome C and myoglobin, less permeable to serum albumin, and almost impermeable to IgG. In the cell attachment test, the results showed that the surface formed by (short-chain alginate)-co-MPEG copolymer could effectively reduce cell adhesion as compared to poly(L-lysine) and alginate. The microcapsules were evaluated by intraperitoneal implantation experiment of mice. The results demonstrated that microcapsules coated with (short-chain alginate)-co-MPEG were more biocompatible than the conventional alginate/PLL/alginate microcapsules.