Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).     

Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (相似文献   

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol)

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.     

Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Surface characterization of poly(ethylene glycol) coated human red blood cells by particle electrophoresis

Recent studies have shown that the covalent attachment of poly(ethylene glycol), abbreviated as PEG, to the surface of human red blood cells (RBC) leads to masking of membrane antigenic sites and inhibition of RBC aggregation. The effects of PEG coating on the regions near the RBC glycocalyx were thus explored using cell micro-electrophoresis. Both linear (3.35, 18.5, 35.0) and an 8-arm 35.9 kDa reactive PEG were used; in one series, thick cross-linked coats were obtained using a branched PEG amine as a cross-linker. The results indicate marked decreases of RBC mobility (up to 90%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment is predominantly to primary amine groups, such decreases of mobility most likely reflect structural changes near and within the RBC glycocalyx rather than decreased surface charge density. Experimental data were analyzed using a theoretical approach which allows calculation of the thickness and friction of the polymer layers: (1) for linear PEGs, thickness increased and friction decreased with polymer mass; (2) compared to linear PEGs of similar molecular mass, thickness was less and friction was greater for the branched PEG; (3) cross-linked PEG coatings were more than 50 nm thick and were insensitive to changes of ionic strength. These observations are consistent with the aggregation behavior of PEG-coated RBC and indicate the usefulness of micro-electrophoresis methods for studies of covalently-attached polymers: the resulting calculated thickness and friction factors should be of value in achieving desired cellular surface characteristics or levels of cell-cell interaction.     

Quantitative Determination of Polyethylene Glycol Based upon Its Salting Out and Partitioning of a Dye into the Resulting Aqueous Two-Phase System

A method is described that allows quantitative determination of polyethylene glycol (PEG) concentrations by spectrophotometric measurement of fluorescein dye absorbance after its partitioning into an aqueous two-phase system containing mPEG (M_r 5 kDa) in the upper phase and ammonium sulfate in the lower phase. The absorbance decrease of fluorescein in the lower phase is directly proportional to the mPEG concentration, with two proportionality constants equal to 4.42 × 10⁵ and 2.84 × 10⁵ M⁻¹ cm⁻¹ in the range of 0-0.4 and 0.4-1 μM, respectively. This experimental technique can be extended to PEGs of other molecular weights by means of calibration curves that give for each size of PEG the adequate proportionality constants. The results indicate that the quantitative determination is not affected by the presence of many substances such as proteins, reducing agents, and salts, at the usual concentrations.     

Conjugation of methoxypolyethylene glycol to the surface of bovine red blood cells

Methoxypolyethylene glycol (mPEG) covalently bound to the surface of human red blood cells (hRBCs) has been shown to decrease immunological recognition of hRBC surface antigens (Bradley et al., 2002). However, there is an increasing shortage of hRBC donations, thus making hRBCs scarce and expensive (Davey, 2004; Riess, 2001). The goal of this study is to similarly PEGylate the surface of bovine RBCs (bRBCs) with the aim of reducing the demand on human blood donations needed for blood transfusions. This study investigates the feasibility of modifying the surface of bRBCs with the succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) for use as a potential blood substitute. The oxygen binding affinity of PEGylated bRBCs was moderately increased with increasing initial SPA-mPEG concentrations up to 4 mM when reacted with bRBCs at a hematocrit of 12%. Oxygen transport simulations verified that SPA-mPEG conjugated bRBCs could still transport oxygen to pancreatic islet tissues even under extreme conditions. PEGylated bRBCs reconstituted to a hematocrit of 40% exhibited viscosities on the order of approximately 3 cp, similar to hRBCs at the same hematocrit. Taken together, the results of this study demonstrate the success of PEGylating bRBCs to yield modified cells with oxygen binding, transport and flow properties similar to that of hRBCs.     

Partitioning of individual flexible polymers into a nanoscopic protein pore

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane beta-barrel of individual protein pores formed from staphylococcal alpha-hemolysin (alphaHL). The interactions of the alpha-hemolysin pore with the PEGs (M(w) 940-6000 Da) fell into two classes: short-duration events (tau approximately 20 micro s), approximately 85% of the total, and long-duration events (tau approximately 100 micro s), approximately 15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Pi) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Pi are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Pi decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than approximately 5 kDa, Pi reached a limiting value suggesting that these PEG chains cannot fit entirely into the beta-barrel.     

Determinants of liposome partitioning in aqueous two-phase systems: Evaluation by means of a factorial design

This article evaluates the influence of five parameters on liposome partitioning in aqueous two-phase systems (ATPSs), composed of poly(ethyleneglycol) (PEG)/dextran (Dx), using the factorial experimental design together with a multiple regression. Mathematical models to quantify the influence of these parameters, individually and/or jointly, on liposome partitioning in ATPS were developed. The models were statistically tested and verified by experimentation. This approach was then used to define the conditions for the preferential accumulation of liposomes in the top PEG-rich phase. The models predicted a significant effect of liposome surface charge, PEG molecular weight, phase-forming polymer concentration, and phosphate ion concentration on the partition behavior of liposomes. For negatively charged liposomes, it was found that the smaller the molecular weight of PEG and polymer concentration and the larger the phosphate ion concentration, the greater the partition coefficient of the liposomes. No significant effect of pH, at the range of 6-8, on liposome partitioning was noted. This approach has led to the development of an optimal two-phase system where 90% of negatively charged liposomes accumulated in the PEG phase. In addition to the general scientific value of this research, it has a technological importance as ATPSs may be useful for removing the unentrapped drug from liposomes during their preparation for pharmaceutical applications. (c) 1996 John Wiley & Sons, Inc.     

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution.     

Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model.

Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.     

Enzymatic hydrolysis of cellulose in aqueous two-phase systems. I. Partition of cellulases from Trichoderma reesei

The partitioning of endo-beta-glucanase, exo-beta-glucanase, and beta-glucosidase from Trichoderma reesei QM 9414 in aqueous two-phase systems has been studied with the object of designing a phase system for continuous bioconversion of cellulose. The partitioning of the enzymes in two-phase systems composed of various water soluble polymeric compounds were studied. Systems based on dextran and polyethylene glycol (PEG) were optimal for one-sidedly partitioning the enzymes to the bottom phase. The influence of polymer molecular weights, polymer concentration, ionic composition of the medium, pH, temperature, and adsorption of the enzymes to cellulose on the enzyme partition coefficients (K) were studied. By combining the effects of polymer molecular weight and adsorption to cellulose, K values could be reduced for endo-beta-glucanase to 0.02 and for beta-glucosidase to 0.005 at 20 degrees C in a phase system of Dextran 40-PEG 40000 in the presence of excess cellulose, At 50 degrees C, K values were increased by a factor of two. In a phase system based on inexpensive crude dextran and PEG, the partition coefficient for endo-beta-glucanase was 0.16 and for beta-glucosidase was 0.14 at 20 degrees C with excess cellulose present.     

Solid-phase PEGylation of recombinant interferon alpha-2a for site-specific modification: process performance, characterization, and in vitro bioactivity

'Solid-phase' PEGylation, in which a conjugation reaction attaches proteins to a solid matrix, has distinct advantages over the conventional, solution-phase process. We report a case study in which recombinant interferon (rhIFN) alpha-2a was adsorbed to a cation-exchange resin and PEGylated at the N-terminus by 5, 10, and 20 kDa mPEG aldehydes through reductive alkylation. After PEGylation, a salt gradient elution efficiently purified the mono-PEGylate of unwanted species such as unmodified IFN and unreacted PEG. Mono-PEGylation and purification were integrated into a single, chromatographic step. Depending on the molecular weight of the mPEG aldehyde, the mono-PEGylation yield ranged from 50 to 65%. Major problems associated with the solution-phase process such as random or uncontrollable multi-PEGylation and post-PEGylation purification difficulties were overcome. N-terminus sequencing and MALDI-TOF mass spectrophometry confirmed that the PEG molecule was conjugated only to the N-terminus. A cell proliferation study indicated reduced antiviral activity of the mono-PEGylate compared to that of the unmodified IFN. As higher molecular weight PEG was conjugated, in vitro bioactivity and antibody binding activity, as measured by a surface plasmon resonance biosensor, decreased. Nevertheless, trypsin resistance and thermal stability were considerably improved .     

A method to optimize PEG-coating of red blood cells

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood group antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH = 8.7, temperature = 14 degrees C, and reaction time = 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface hydrophobicity

Two different series of hydrophobically modified proteins were partitioned in a number of aqueous two-phase systems (ATPS) to investigate the effect of hydrophobicity as a single property on partitioning. The modified proteins were derived from beta-lactoglobulin and bovine serum albumin (BSA). Measurement of the surface hydrophobicity of the proteins is important; hydrophobic interaction chromatography (HIC) was used for this purpose. The resolution of the systems (R) in terms of protein surface hydrophobicity and the intrinsic hydrophobicity (log P(0)) of the systems was established. The effect of the addition of NaCl to PEG/phosphate and PEG/dextran systems was analyzed in terms of the hydrophobicity difference between the phases and their ability to promote hydrophobic interactions between the protein surface and the PEG molecules. The values for R and log P(0) differed somewhat depending on which group of modified proteins was used for partitioning. The addition of NaCl to PEG/phosphate systems promoted an increase in the values of R, showing an important effect on the resolution of the systems for protein surface hydrophobicity (twice as high when compared with systems without NaCl). For PEG/dextran systems, the addition of 9% NaCl (w/w) promoted an improvement in the resolution toward surface hydrophobicity with an increase of 60% on the value of R. (c) 1996 John Wiley & Sons, Inc.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Role of the methoxy group in immune responses to mPEG-protein conjugates

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface charge

A series of charge-modified thaumatins with different values of surface charge were partitioned in aqueous two-phase systems (ATPS) to study the effect of surface charge as a single property on partitioning. Electrophoretic mobility of the proteins in titration curves was used as a measure of surface charge. Four modified proteins derived from thaumatin with the following values of isoelectric point: 8.70, 8.15, 5.60, and 4.50 were used for partitioning. The resolution of the systems in terms of protein surface charge was calculated. Partitioning of modified thaumatins in PEG 4000/dextran systems with phosphate buffer, Tris buffer, NaCl, KCl, and sulfate salts was carried out. Among the sulfate salts tested, the addition of 50 mM Li(2)SO(4) to the system buffered with phosphate gave the highest value of resolution for differences in surface protein charge (RSPC). It shows a decrease in the value of K (partition coefficient) with an increase in the protein's charge. The addition of 100 mM KCl to the system promoted the opposite effect on the RSPC value. Charge-modified proteins were partitioned in PEG/salt systems to investigate the ability of these systems for resolving differences in surface charge. The PEG/citrate system seemed to have almost no ability for resolving proteins on the basis of surface charge differences; PEG/phosphate systems had some capability for resolving differently charged proteins. The more negative proteins tended to have higher values of K than the more positively charged fractions. The use of charge-modified proteins allowed the investigation of the effect of protein surface charge on partitioning in aqueous two-phase systems independently from other protein parameters as they were prepared from a common parent protein thaumatin. This technique provides an interesting novel tool to investigate the effect of protein surface charge on partitioning in ATPS taking protein charge as an independent parameter. (c) 1996 John Wiley & Sons, Inc.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Inhibition of polymer-induced red blood cell aggregation by poloxamer 188

Previous reports have suggested that non-ionic poloxamer surfactants of appropriate molecular mass and composition can reduce red blood cell (RBC) aggregation in whole blood and in RBC-plasma suspensions. We have thus evaluated this phenomenon for RBC aggregated by several water-soluble polymers, using poloxamer 188 (P188), a non-ionic, tri-block molecule (total molecular mass of 8.40 kDa, 80% polyoxyethylene). Human RBC were washed, then re-suspended in isotonic solutions of dextran 70 (70.3 kDa), dextran 500 (476 kDa), PVP (360 kDa) or P-L-GLU (61.2 kDa); density-separated RBC were also studied. RBC aggregation was quantitated via a computerized Myrenne Aggregometer (extent, strength) and by the Microscopic Aggregation Index (MAI) method. Over the range of 0.5 to 5 mg/ml, poloxamer 188 inhibited both the extent and strength of aggregation in a dose-dependent manner, with the magnitude of the decrease related to polymer type (e.g., at 5 mg/ml, 62% decrease for dextran 70 vs. 14% decrease for P-L-GLU); MAI results with dextran 70 also showed a dose-dependent decrease. Poloxamer 188 at 5 mg/ml was more effective with younger, less-dense cells. Based upon the depletion model for polymer-induced aggregation, these findings suggest that poloxamer 188 acts by penetrating the depletion layer near the glycocalyx, thereby reducing the osmotic gradient between the intercellular gap and the suspending medium. Regardless of the specific mechanism(s) of action, poloxamers appear to offer interesting approaches for future basic science and clinical studies, and thus the possibility for greater insight into RBC aggregation.