Modulation of red blood cell aggregation and blood ... - CiteSeerX

Biorheology 38 (2001) 239–247 IOS Press

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Modulation of red blood cell aggregation and blood viscosity by the covalent attachment of Pluronic copolymers Jonathan K. Armstrong ∗ , Herbert J. Meiselman, Rosalinda B. Wenby and Timothy C. Fisher Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 29 January 2001 Accepted in revised form 28 February 2001 Abstract. Despite many years of research, the physiologic or possible pathologic significance of RBC aggregation remains to be clearly determined. As a new approach to address an old question, we have recently developed a technique to vary the aggregation tendency of RBCs in a predictable and reproducible fashion by the covalent attachment of nonionic polymers to the RBC membrane. A reactive derivative of each polymer of interest is prepared by substitution of the terminal hydroxyl group with a reactive moiety, dichlorotriazine (DT), which covalently bonds the polymer molecule to membrane proteins. Pluronics are block copolymers of particular interest as these copolymers can enhance or inhibit RBC aggregation. Pluronics exhibit a critical micellization temperature (CMT): a phase transition from predominantly single, fully hydrated copolymer chains to micelle-like structures. The CMT is a function of both copolymer molecular mass and concentration. This micellization property of Pluronics has been utilized to enhance or inhibit RBC aggregation and hence to vary low-shear blood viscosity. Pluroniccoated RBCs were prepared using reactive DT derivatives of a range of Pluronics (F68, F88, F98 and F108) and resuspended in autologous plasma at 40% hematocrit. Blood viscosity was measured at a range of shear rates (0.1–94.5 s−1 ) and at 25 and 37◦ C using a Contraves LS-30 couette low shear viscometer. RBC aggregation and whole blood viscosity was modified in a predictable manner depending upon the CMT of the attached Pluronic and the measurement temperature: below the CMT, RBC aggregation was diminished; above the CMT it was enhanced. This technique provides a novel tool to probe some basic research questions. While certainly of value for in vitro mechanistic studies, perhaps the most interesting application may be for in vivo studies: typically, intravital experiments designed to examine the role of RBC aggregation in microvascular flow require perturbation of the suspending plasma to promote or reduce aggregation (e.g., by the addition of dextran). By binding specific Pluronics to the surface, we can produce RBCs that intrinsically have any desired degree of increased or decreased aggregation when suspended in normal plasma, thereby eliminating many potential artifacts for in vivo studies. The copolymer coating technique is simple and reproducible, and we believe it will prove to be a useful tool to help address some of the longstanding questions in the field of hemorheology. Keywords: Red blood cell, aggregation, Pluronic, poloxamer, micellization, covalent

1. Introduction Studies designed to investigate the effects of red blood cell (RBC) aggregation on blood rheology or flow often require the use of aggregation-inducing macromolecules (e.g., dextrans 70 or 500 kDa, poly(ethylene oxide) 35 kDa, polyvinylpyrrolidone 360 kDa) [9,13,15,31,32]. These water-soluble polymers are either dissolved in the suspending phase for in vitro RBC studies, or infused as concentrated * Address for correspondence: Dr. Jonathan K. Armstrong, Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, 1333 San Pablo Street, Los Angeles, CA 90033, USA. Tel.: +1 323 442 3387; Fax: +1 323 442 1617; E-mail: [email protected].

0006-355X/01/$8.00  2001 – IOS Press. All rights reserved

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J.K. Armstrong et al. / RBC aggregation modulated by Pluronic copolymers

stock solutions when used for in vivo measurements [13,15,32]. However, interpretation of experimental results using this approach may be complicated by alteration of the suspending phase viscosity and dilution of plasma components (e.g., fibrinogen) by the added or infused agent. A method to induce or inhibit red cell aggregation in a controlled and predictable manner by modification of intrinsic RBC properties, without alteration of the plasma phase, would obviously be advantageous. Pluronic copolymers, also known as poloxamers, are block copolymers composed of a central hydrophobic block of poly(propylene glycol) (PPG) flanked by two hydrophilic poly(ethylene glycol) (PEG) chains [8] (Fig. 1). Pluronics are used industrially as surfactants and are available in a range of molecular masses and PEG/PPG ratios (e.g., 1–14 kDa, 10–80% PEG). In aqueous solution, Pluronics exhibit a critical micellization temperature (CMT): a phase transition from predominantly single, fully hydrated copolymer chains to micelle-like structures [2,3,10]. For many surfactant compounds and polymers (e.g., sodium dodecyl sulfate), a critical micelle concentration (CMC) is observed which is temperature independent. However, since the self-association behavior of Pluronic copolymers is significantly affected by temperature, and given that all of the commercially available Pluronics exist predominantly in a single chain form in aqueous solutions at temperatures below 10◦ C, it is practical to express the micellization behavior as a function of temperature (CMT). The micellization is consequent upon dehydration and associative interactions between the hydrophobic PPG blocks of the copolymer chains; the micelles remain in solution due to the hydrated hydrophilic PEG chains surrounding the PPG core. The CMT for each Pluronic is dependent on copolymer concentration [2,3] and inversely dependent upon the molecular mass of the PPG block [10]; those with larger PPG blocks micellize at lower temperatures. High sensitivity differential scanning calorimetry [11,14,34], has been employed to determine the CMT values for several Pluronic copolymers dissolved in plasma [4], these data are shown in Table 1. When added to plasma, Pluronic copolymers inhibit or promote RBC aggregation depending on the temperature of the sample and the CMT of the particular Pluronic added: below the CMT, RBC aggregation is inhibited, whereas above the CMT, promotion of RBC aggregation is observed [4,5]. We thus postulated that control of RBC aggregation could be achieved by the covalent attachment of carefully selected Pluronics to the RBC membrane [16]. Below the CMT, covalently attached Pluronics should not self-associate and should inhibit RBC aggregation due to polymer–polymer steric repulsion. Conversely, above the CMT,

Fig. 1. Generalized structure of a Pluronic copolymer (poloxamer), an ABA block copolymer comprised of a central block of poly(propylene glycol) flanked by two poly(ethylene glycol) chains. Table 1 Critical micellization temperatures (CMT) of pluronic copolymers in human plasma∗ Pluronic Molecular mass (g mol−1 ) CMT in plasma (◦ C) F68 8400 48 F88 11400 37 F98 13000 30 F108 14600 23 ∗ All studies were performed at a plasma concentration of 5 mg ml−1 . All Pluronic copolymers shown here contain 80% PEG (w/w).


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the copolymers will attempt to micellize, and aggregation of RBC should be significantly enhanced due to the associative interactions of Pluronics between adjacent cells. To facilitate covalent attachment of the Pluronic to the surface of a RBC, the terminal hydroxyl group of the polymer chain must first be converted to a reactive intermediate. Reactive derivatives of poly(ethylene glycol) (PEG) have been demonstrated to effectively coat RBCs resulting in reduced RBC aggregation and whole blood viscosity [6], reduced antigenicity [6,17,22], and the prevention of malarial parasitic invasion of RBCs by Plasmodium falciparum [12]. In these studies the reactive moiety of the Pluronic derivatives, dichlorotriazine, was selected on the basis of its apparent short aqueous half-life at a pH > 7.0: sustained reactivity of the polymer derivative would be expected to yield a cross-linked RBC mass following the first centrifugation after incubation with the reactive derivative, yet no such artifact was evident. We have used the same methodology as previously described for PEG [1,7,12,17] for the preparation of reactive dichlorotriazine intermediates of Pluronics (see below). Herein we present the effects on RBC aggregation and blood viscosity for a series of Pluronic copolymers covalently attached to the surface of RBCs; data are shown as a function of copolymer molecular mass and temperature. The potential applications of this technique for in vivo studies are also discussed. 2. Materials and methods 2.1. Chemicals Pluronic copolymers (poloxamers) F68, F88, F98 and F108 were a gift from BASF Performance Chemicals (Parsippany, NJ). The total molecular mass of each of these copolymers is shown in Table 1; note each of these Pluronics contained 80% PEG on a mass basis [8]. Trichlorotriazine, anhydrous sodium carbonate, benzene and cyclohexane were purchased from Sigma Chemical Company (St. Louis, MO). Benzene and cyclohexane were distilled over sodium metal prior to use. 2.2. Pluronic derivatization Preparation of reactive dichlorotriazine intermediates of Pluronics [Pluronic-DT, (Cl2 N3 C3 )-PEGPPG-PEG-(C3N3 Cl2 )] was performed using a modification of the method described by Abuchowski et al. [1]. Briefly, vacuum-dried Pluronic was dissolved in hot anhydrous benzene, cooled to 15◦ C, and then slowly added to a solution of trichlorotriazine (25 fold molar excess) in benzene. Anhydrous sodium carbonate was then added as a catalyst for the reaction. The mixture was stirred for 24 hours (F68, F88) or 48 hours (F98, F108) at 15◦ C under an atmosphere of dry nitrogen, then filtered or centrifuged at 1750 × g for 10 minutes to remove the sodium carbonate. The Pluronic-DT was then precipitated with an excess of anhydrous cyclohexane, collected by filtration, resuspended and washed and filtered a further 5 times in cyclohexane to remove any unreacted trichlorotriazine. The Pluronic-DT derivatives were dried under a stream of dry nitrogen for 24 hours at room temperature and stored at −20◦ C under nitrogen until use. 2.3. Blood preparation Blood was drawn into EDTA (1.5 mg ml−1 ) by sterile venipuncture from healthy donors after informed consent, washed twice with isotonic 10 mM Dulbecco’s PBS (pH 7.4, 285 mOsmkg−1 ), and then resuspended at 50% hematocrit (hct) in 30 mM triethanolamine–NaCl buffer (pH 8.60, 295 mOsmkg−1 ).

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A fresh stock solution of Pluronic-DT was prepared in ice-cold acidified saline (0.9% NaCl containing 5 mM HCl) and was added to the RBC suspension at a concentration of 5 mg of reactive polymer per ml of packed RBC, yielding a suspension concentration of 2.5 mg ml−1 . The blood samples were mixed gently at 10◦ C for 1 hour, following which the RBC were washed twice with PBS and then re-suspended at 40% hematocrit in autologous plasma. The Pluronic-coating process was performed at 10◦ C, below the CMT, to ensure that the reactive copolymer was predominantly in a single chain state and to prevent the formation of “permanent” covalently cross-linked aggregates. At room temperature, reducing the incubation hct below 50% did not prevent the covalent cross-linking of RBCs, whereas lowering the incubation temperature below the CMT adequately produced polymer-coated single RBCs. Control (i.e., uncoated) RBCs were washed twice in PBS, then re-suspended in autologous plasma at 40% hct. The morphology of all RBC samples was examined by differential interference contrast (DIC) light microscopy using dilute wet-mounts prepared with autologous plasma. 2.4. Viscosity measurements The apparent viscosity of the RBC suspensions at 25 and 37◦ C was measured over a range of shear rates (0.1–94.5 s−1 ) using a Contraves LS-30 Couette viscometer. At low shear rates where aggregating suspensions exhibit torque decay at constant shear, an extrapolation to zero time method was employed to obtain the correct torque value [30].

3. Results Figure 2 presents apparent viscosity-shear rate data, at both 25 and 37◦ C, for Pluronic-coated and control (i.e., uncoated) RBCs suspended in autologous plasma at a 40% hematocrit. As anticipated [27,30], control RBCs in plasma exhibit marked non-Newtonian flow behavior, with apparent viscosity increasing as the shear rate is decreased. This shear-rate dependence of viscosity is a classical observation in the field of hemorheology, and it has been clearly established that increased low-shear viscosity is a direct result of the formation of RBC aggregates (e.g., rouleaux) and the development of a three-dimensional aggregate structure at low shear and at stasis [27,30]. As shown in Fig. 2, the rheological effects of covalently coating RBCs with a Pluronic depend on the critical micellization temperature (CMT) of the copolymer. PLURONIC-F68: Pluronic F68-coated RBCs at 25◦ C showed nearly Newtonian flow behavior and markedly reduced low shear viscosity versus control (high shear viscosity = 5.7 and 5.6 mPa.s, low shear viscosity = 10.2 and 71.1 mPa.s, respectively), indicating that the Pluronic coating substantially reduced or abolished RBC aggregation. At 37◦ C, low shear viscosity was 33.0 mPa.s for coated cells versus 55.8 mPa.s for control, indicating a relative reduction, but not complete inhibition, of RBC aggregation. PLURONIC-F88: Pluronic F88-coated RBCs at 25◦ C showed flow behavior similar to control (low shear viscosity = 72.8 mPa.s), indicative of a similar degree of RBC aggregation in the control and treated samples. However, at 37◦ C, the low shear viscosity (158 mPa.s) was significantly higher than control, indicating an enhancement of RBC aggregation at the higher temperature. PLURONIC-F98 and PLURONIC-F108: Pluronic F98- and F108-coated RBCs showed greatly increased low shear blood viscosity at both temperatures (269 and 277 mPa.s at 25◦ C; 232 and 246 mPa.s at 37◦ C for F98 and F108, respectively), indicating greatly increased RBC aggregation. Note also that


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Fig. 2. Viscosity-shear rate data for RBCs suspended in autologous plasma at 25 and 37◦ C. Control (uncoated) RBCs () shows the typical non-Newtonian behavior for an aggregating system, the increase in viscosity with decreasing shear rate indicative of RBC aggregation. Newtonian behavior is observed for F68-coated RBCs () at 25◦ C indicative of no aggregation, and reduced low shear blood viscosity at 37◦ C in autologous plasma relative to control. F88-coated RBCs () show neutral behavior at 25◦ C and an increase in low shear blood viscosity at 37◦ C. F98 () and F108 ()-coated RBC show a significant increase in low shear blood viscosity at 25 and 37◦ C indicative of enhanced RBC aggregation.

for the F98 and F108 coating, the effects of enhanced RBC aggregation persist over a relatively wide range of shear and remain evident up to at least six inverse seconds. Pluronics possess no chromophores and there are no simple assays to determine the exact concentration of copolymer covalently bound to the surface of RBCs without the use of a radiolabeled copolymer. However, preliminary results (not shown) have been obtained using an indirect approach to determine the concentration of copolymer remaining in solution. In brief, gel permeation chromatography data (2×PLAquagel OH-30 columns, 10 mM phosphate buffer pH 7.4, flow rate = 1 ml min−1 , 1000 psi, ambient temperature, refractive index detector) indicate that