Determination of pore diameter and molecular weight cut-off of hydrogel-membrane liquid-core capsules for immunoisolation

The exclusion limit expressed as the largest pore size of capsules composed of hydrogel Ca-alginate membrane and hydroxy-propyl-ammonium starch liquid core considered as the immunoprotective system has been determined by means of inverse size exclusion chromatography with dextran molecular weight standards. The exclusion limits of the capsules were not influenced by the change of starch concentration in the core solution from 4 to 6% but were influenced by the change in alginate concentration in the membrane from 0.5 to 1.0%, causing the membranes to be less permeable. It was found that the diameter of the largest pores in hydrogel membranes was in the range 7.2-8.0 nm. Based on the relationship between solute size and its molecular weight, the capsules had an approximate exclusion limit of 21-25 kD for dextran and 78-103 kD for protein, which is sufficient to block the antibodies penetrating through the membrane.     

In vivo biocompatibility evaluation of cellulose macrocapsules for islet immunoisolation: Implications of low molecular weight cut-off

Earlier we showed the in vitro suitability of cellulose molecular dialysis membrane of low cut-off for islet immunoisolation. Using a syngenic islet transplantation model, here we report the in vivo biocompatibility status and discuss implications of the low molecular weight cut-off of this membrane. Streptozotocin diabetic mice were transplanted ip with islet grafts in cellulose macrocapsules and monitored for 6 weeks for their blood glucose profiles. Membrane biocompatibility was evaluated by assessing islet graft morphometry, viability, functionality, and tissue reaction against the capsules. Animals (n = 10) attained normoglycemia after 1 week of transplantation and remained nondiabetic throughout a follow-up of 6 weeks. Animals subjected to a glucose tolerance test at the end of the study showed delayed glycemic control, indicating a delayed insulin response. Grafts retrieved after the 15(th) day and after 6 weeks showed viable islets, between 70-80% and approximately 40%, respectively, with morphometric parameters similar to freshly isolated islets. Graft retrievals resulted in a recurrence of hyperglycemia, indicating a functional tissue mass. A mild tissue reaction with a few immunocytes and a very thin fibrous capsular reaction were seen against the macrocapsule membrane. This study points out the in vivo suitability of cellulose membrane for islet immunoisolation. However, applicability of this membrane is limited due to its low cut-off value. Membranes with a higher molecular weight cut-off may render better glycemic control and higher graft viability.     

Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads

The enzyme bovine carbonic anhydrase (BCA) has been immobilized in the chitosan-alginate system for the first time, to catalyze the conversion of CO₂ to HCO₃-. Chitosan-coated alginate beads are a biodegradable and environmentally benign matrix, chosen for application of the enzyme in a novel biomimetic CO₂ sequestration system. The feasibility of the system and immobilization of the enzyme were demonstrated in our earlier studies [1–3]. Optimization of the matrix to improve the retention time of the enzyme in an encapsulated form is the subject of the present study. The improvement in the molecular weight cut-off of the beads was accomplished by adjusting the crosslinking conditions, coating composition, and molecular weight of the system. The quantity of enzyme released from the system was measured by a Bio-Rad protein assay. Poly-L-lysine was also used as a coating reagent for comparison purposes. The presence of a coating on the alginate beads was verified by Kjeldahl analyses. The difference in the microstructures of alginate and chitosan/alginate beads was demonstrated by SEM studies. Mineralization of the chitosan/alginate matrix in the presence of CaCO₃ was also studied by FT-IR, to assess the possibility of using the beads continuously in a bioreactor.     

Matrix molecular weight cut-off for encapsulation of carbonic anhydrase in polyelectrolyte beads

The enzyme bovine carbonic anhydrase (BCA) has been immobilized in the chitosan-alginate system for the first time, to catalyze the conversion of CO2 to HCO3-. Chitosan-coated alginate beads are a biodegradable and environmentally benign matrix, chosen for application of the enzyme in a novel biomimetic CO2 sequestration system. The feasibility of the system and immobilization of the enzyme were demonstrated in our earlier studies. Optimization of the matrix to improve the retention time of the enzyme in an encapsulated form is the subject of the present study. The improvement in the molecular weight cut-off of the beads was accomplished by adjusting the cross-linking conditions, coating composition, and molecular weight of the system. The quantity of enzyme released from the system was measured by a Bio-Rad protein assay. Poly-L-lysine was also used as a coating reagent for comparison purposes. The presence of a coating on the alginate beads was verified by Kjeldahl analyses. The difference in the microstructures of alginate and chitosan/alginate beads was demonstrated by SEM studies. Mineralization of the chitosan/alginate matrix in the presence of CaCO3 was also studied by FT-IR, to assess the possibility of using the beads continuously in a bioreactor.     

Determination of molecular weight distributions by gel-permeation chromatography

The theoretical basis for the determination of molecular weight distributions of polymers by gel-permeation chromatography has been investigated, using the previously developed theory of partition chromatography. Expressions determining the position and broadening of the concentration distribution in the elution curve have been deduced and the molecular weight dependence of the parameters characterizing the process has been considered. A method is proposed for determining the molecular weight distribution by a convolution procedure.     

Determination of the molecular weight of bovine enterovirus RNA by nuclease digestion.

The mol. wt. of a [32P]-labelled bovine enterovirus RNA has been determined by digesting with pancreatic RNase and separating the resulting oligonucleotides using a two-stage fractionation method on DEAE-Sephadex in 7 M-urea at pH 7-6 and pH 3-0. We have estimated the number of nucleotides as 8612 plus or minus 55. This corresponds to a mol. wt. of 2-93 plus or minus 0-02 X 10-6 which is in aggrement with estimates obtained by sedimentation and gel electrophoresis techniques.     

Alginate-based microcapsules for immunoisolation of pancreatic islets

Transplantation of microencapsulated cells is proposed as a therapy for the treatment of a wide variety of diseases since it allows for transplantation of endocrine cells in the absence of undesired immunosuppression. The technology is based on the principle that foreign cells are protected from the host immune system by an artificial membrane. In spite of the simplicity of the concept, progress in the field of immunoisolation has been hampered for many years due to biocompatibility issues. During the last years important advances have been made in the knowledge of the characteristics and requirements capsules have to meet in order to provide optimal biocompatibility and survival of the enveloped tissue. Novel insight shows that not only the capsules material but also the enveloped cells should be hold responsible for loss of a significant portion of the immunoisolated cells and, thus, failure of the grafts on the long term. Microcapsules without cells can be produced as such that they remain free of any significant foreign body response for prolonged periods of time in both experimental animals and humans. New approaches in which newly discovered inflammatory responses are silenced bring the technology of transplantation of immunoisolated cells close to clinical application.     

Determination of molecular weight, isoelectric point, and glycoprotein moiety for the principal skin test-reactive component of histoplasmin.

A purified component designated HPD (histoplasmin-purified derivative) dII was isolated from two different cru-e histoplasmin lots by a combination of gel filtration and polyacrylamide disc electrophoresis (Sprouse, 1969). A 0.05-mug portion of HPD dII was reactive and specific in detection of delayed hypersensitivity in guinea pigs experimentally infected with Histoplasma capsulatum. The objective of this study was to characterize this skin test-reactive component for (i) homogeneity, (ii) molecular weight, (iii) isoelectric point, and (iv) composition. Sephadex chromatography, polyacrylamide disc electrophoresis, sucrose density gradient ultracentrifugation, acid and heat denaturation, and immunoelectrophoresis indicate that HPD dII is (i) homogeneous, (ii) of approximately 12,000 molecular weight, and (iii) a glycopeptide of approximately 60% carbohydrate and 40% proteinaceous composition. The marked acid and heat stability exhibited by the compound probably is attributable to the prominent carbohydrate moiety in the molecule. Isoelectric focusing indicated an isoelectric point of 5.68. This would suggest that dII is an acidic compound with either predominance of acidic amino acid residues in the molecule or, more probably, an abundance of electron donors in the carbohydrate moiety. In summary, HPD dII appears to be a glycopeptide of approximately 12,000 molecular weight, reactive in elicitation of delayed hypersensitivity of histoplasmosis.     

Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material.

The patterns of DNA degradation in frozen, methanol-fixed, and formalin-fixed tissues were investigated by high-performance liquid chromatography (HPLC). The chromatograms all yielded one major peak with or without several extra minor peaks representing molecular weights of preserved genomic DNA. The most characteristic differences were in the retention times of the major peaks, with the earliest major peak occurring in the formalin-fixed tissues, and followed by the methanol-fixed, and frozen tissue samples, in that order. This means that the molecular weight of the DNA from formalin-fixed tissue is much shortened than that recovered from methanol-fixed tissue and frozen tissue. The results also indicated that a small amount of higher molecular weight DNA is still preserved in formalin-fixed tissues. To improve the amplification efficiency of polymerase chain reaction (PCR) analysis of formalin-fixed material, we isolated the higher molecular weight DNA from formalin-fixed, paraffin-embedded tissue from four different organs and compared the amplification efficiencies with those of the crude DNA extract. We used eight sets of oligonucleotide primers producing 262 to 989 base pair (bp) fragments of beta-globin. The results showed that the PCR amplification analyses were more efficient with the isolated higher molecular weight DNA than with the crude DNA extract. Our study demonstrated that not all the DNA in formalin-fixed, paraffin-embedded tissue samples is totally degraded but that a small amount of higher molecular weight DNA persists. The feasibility of molecular diagnosis using formalin-fixed material can be improved by isolating the preserved higher molecular weight DNA by HPLC.     

Isolation and characterization of low molecular weight antibodies for Treponema pallidum.

A low molecular weight antibody which coated Treponema pallidum without producing any apparent reaction was isolated from human syphilitic serum. Its electrophoretic mobility was that of IgG immunoglobulins, and molecular weight was between 115,000 and 120,000.     

Intrinsic viscosity-molecular weight relationship for low molecular weight cellulose and oligosaccharides

The viscosity behaviour of low molecular weight cellulose and oligosaccharides has been investigated. In the high molecular weight region the double-logarithmic plot of intrinsic viscosity vs molecular weight is linear down to approximately DP = 150. On passing to lower molecular weights the slope of the curve at first increases, passes through a maximum and finally decreases to very low values in the oligomere region.     

A simple method for quantifying IgM of low molecular weight.

A new and simple method for quantitation of IgM of low molecular weight in sera by radial immunodiffusion in 7% agar gel is described. The test is easy to perform and permits exact determination of low molecular weight IgM concentration down to very small quantities (1 mg%). Results obtained with the method described agree with previously used assay procedures and offer the advantage of convenience and rapidity.     

Antigenicity of low molecular weight surfactant species.

The authors tested the antigenicity of human lung surfactant isolated from amniotic fluid. Mice and rabbits were immunized. Rabbit polyclonal antisera to these surfactant preparations were absorbed with normal human plasma proteins. Polyclonal antisera reacted with both high molecular weight (35 kd) surfactant apoprotein and to lower molecular weight species, both 18 kd and 9 kd. Mice were used to generate monoclonal antibodies to surfactant. Enzyme-linked immunosorbant assay was used to identify five monoclonal antibodies that reacted with surfactant. By Western blot analysis, all of these recognized a low molecular weight surfactant species (9 kd) that could be either SP-B or SP-C. One reacted with a 37 kd protein in the surfactant preparation, consistent with SP-A. One monoclonal antibody also recognized a higher molecular weight species (44 kd) of unknown origin. The ability of antisera and monoclonal antibodies to inhibit the functional activity of surfactant was assayed using a pulsating bubble surfactometer. Rabbit polyclonal antisera inhibited initial surface adsorption to equilibrium surface tension and increased the minimum surface tension after 1 and 5 minutes of initiation of pulsations. This inhibitory activity of the antisera was noted in divalent F(ab')2 fragments. Monovalent F(ab) fragments and control normal rabbit sera did not inhibit surfactant function in this assay. Of the anti-surfactant monoclonal antibodies that reacted with surfactant by ELISA and Western blot, three inhibited its capacity to lower surface tension on the pulsating bubble apparatus. The other two monoclonal antibodies showed no functional inhibitory activity. It is concluded that both the 35 kd SP-A and the 9 kd proteins of human surfactant are highly immunogenic and partially crossreactive. Resulting antibodies could alter the ability of surfactant to perform its physiologic function, ie, to lower surface tension.     

Control of molecular weight and molecular weight distribution in ethylene polymerization with metallocene catalysts

The molecular weight (MW) and the molecular weight distribution (MWD) of polyethylene (PE) prepared with various metallocene catalysts were investigated. The weight-average molecular weight (M _w) of PE prepared with dicyclopentadienyltitanium dichloride/aluminoxane (Cp₂TiCl₂/MAO) was about 4,0 · 10⁵, while that of PE obtained with dicyclopentadienylzirconium dichloride/aluminoxane (Cp₂ZrCl₂/MAO) is about 1,0 · 10⁵. PE of bimodal MWD was obtained with the mixed catalyst Cp₂TiCl₂/Cp₂ZrCl₂. Narrow MWD PE was obtained with the mixed catalyst Cp₂ZrCl₂/Et(ind)₂ZrCl₂, because each catalyst produced an M _w of 1,0 · 10⁵. However, the change of temperature during the polymerization with the mixture Cp₂ZrCl₂/Et(ind)₂ZrCl₂ produced PE with a bimodal MWD and a value of the ratio of weight- to number-average molecular weights of M _w/M _n = 4.     

高效凝胶色谱法测定多花黄精多糖分子量与分子量分布

目的：建立多花黄精多糖的分子量与分子量分布分析方法。方法：应用高效凝胶渗透色谱法，色谱柱为Shodex OHPak SB-803HQ（8mm×300mm），流动相为0．71％硫酸钠溶液（内含0．02％叠氮钠），柱温：35℃，示差折光检测器（检测器温度35℃），流速0．5ml·min^-1。结果：根据建立的方法测定，得到多糖的高效凝胶色谱图，计算出多花黄精多糖的重均分子量及其分布。结论：所用方法简便、快速、准确，可用于多花黄精多糖的质量控制。     

Reduced allergenicity of high molecular weight ragweed polymers.

Polymerized ragweed antigens of different molecular weight ranges were studied to determine if the degree of allergenicity was dependent on molecular size of the polymers. In this study, allergenicity is defined as the ability to elict IgE-mediated skin reactivity. Ragweed antigen treated with glutaraldehyde was fractionated into one preparation with a molecular weight range of 200,000 to 20,000,000 and another with molecular weights less than 200,000. Except for the difference in molecular sizes of the two molecular sizes of the two preparations, all ragweed polymers had been treated in an identical fashion. The low molecular weight fraction had cutaneous endpoint reactivity greater than 10(5) to 10(8) times that of the high molecular weight materials. In these and other studies, immunogenicity defined as the ability to induce an IgG antibody response was retained by the high molecular weight material. The results are consitent with the hypothesis that allergenicity decreases as the molecular weight of the polymerized allergen increases.     

Inhibition of phagocytosis by high molecular weight hyaluronate.

The effect of sodium hyaluronate on phagocytosis was studied using a sensitive polystyrene latex sphere assay in mouse peritoneal macrophage monolayers. Viscous solutions of high molecular weight hyaluronate (4.6 X 10(5)--2.8 X 10(6)) caused a dose-dependent inhibition of phagocytosis, but low molecular weight hyaluronate (9.0 X 10(4)) was not inhibitory at equivalent viscosity. The inhibitory effect of high molecular weight hyaluronate did not appear to be mediated by the polyanionic charge of the molecule since sulphated glycosaminoglycans with greater charge density (heparin and chondroitin sulphate) were ineffective. In addition, competitive inhibition studies indicated that a direct effect on possible cell surface membrane receptors was unlikely. Instead, physical factors such as steric hindrance by the continuous polymeric network, were considered of more importance. Alternatively, the hydrophilic polysaccharide may have inhibited phagocytosis by providing an unsuitable surface for adhesive contact between the latex beads and the cell surface.     

Synthesis and molecular weight characterization of low molecular weight end-functionalized poly(4-acryloylmorpholine)

New amphiphilic oligomers ending at one side with a reactive function were synthesized by radical polymerization of a vinyl monomer, N-acryloylmorpholine, in the presence of functionalized chain-transfer agents (CTAs). The oligomers were characterized in terms of molecular weight and molecular weight distribution, by means of analytical size-exclusion chromatography (SEC) working in buffered aqueous media. This has been accomplished by determining a calibration curve for a set of SEC columns, with poly(N-acryloylmorpholine) standards purposely obtained by means of preparative SEC. The transfer constant C_T of some CTAs towards N-acryloylmorpholine has been estimated to be approximately 1.