Research Paper
Effects of amphiphilic star-shaped poly(ethylene glycol) polymers with a cholic acid core on human red blood cell aggregation

https://doi.org/10.1016/j.jmbbm.2012.11.008Get rights and content

Abstract

Elevated red blood cell (RBC) aggregation increases low-shear blood viscosity and is closely related to several pathophysiological diseases such as atherosclerosis, thrombosis, diabetes, hypertension, cancer, and hereditary chronic hemolytic conditions. Non-ionic linear polymers such as poly(ethylene glycol) (PEG) and Pluronic F68 have shown inhibitory effects against RBC aggregation. However, hypersensitivity reactions in some individuals, attributed to a diblock component of Pluronic F68, have been reported. Therefore, we investigated the use of an amphiphilic star-shaped PEG polymer based on a cholic acid core as a substitute for Pluronics to reduce RBC aggregation. Cholic acid is a natural bile acid produced in the human liver and therefore should assure biocompatibility. Cholic acid based PEG polymers, termed CA(PEG)4, were synthesized by anionic polymerization. Size exclusion chromatography indicated narrow mass distributions and hydrodynamic radii less than 2 nm were calculated. The effects of CA(PEG)4 on human RBC aggregation and blood viscosity were investigated and compared to linear PEGs by light transmission aggregometry. Results showed optimal reduction of RBC aggregation for molar masses between 10 and 16 kDa of star-shaped CA(PEG)4 polymers. Cholic acid based PEG polymers affect the rheology of erythrocytes and may find applications as alternatives to linear PEG or Pluronics to improve blood fluidity.

Introduction

Organ and tissue perfusion strongly depends on adequate blood supply to the microcirculation, and subtle disturbances of microcirculatory flow can lead to clinical disorders including tissue dysfunction or ischemia (Rad and Neu, 2009). The hyper viscosity syndrome (i.e., greatly elevated blood viscosity) is a condition associated with enhanced red blood cell (RBC) aggregation that can reduce blood flow and lead to localized stagnation. Increased RBC aggregation has been observed in this condition and is implicated in the pathophysiology of numerous diseases with circulatory disorders such as cardiovascular diseases, chronic and acute inflammatory diseases, diabetes, cancers, sickle cell disease, thalassemia and trauma (MacRury et al., 1993, Ziegler et al., 1994, Shiga et al., 1990). Moreover, epidemiological studies have recognized RBC aggregation as a strong cardiovascular risk factor (Somer and Meiselman, 1993).

Over the past several decades, there has been a strong interest in possible therapeutic agents that can counter RBC hyper-aggregation. For clinical therapy, polymers added to blood to improve rheology by reducing RBC aggregation were proposed (Chien and Jan, 1973, Armstrong et al., 2004). Therapeutic approaches have included infusion of selected polymers or their covalent linkage to the RBC membrane. These polymers include linear poly(ethylene glycol) (PEG), and an amphiphilic block copolymer of poly(propylene glycol) (PPG) and PEG (generic name poloxamers, trademark Pluronics and Lutrol); the block copolymers are comprised of a central hydrophobic moiety core of PPG flanked by two equal PEG chains, PEG–PPG–PEG (Toth et al., 2000, Armstrong et al., 1995, Armstrong et al., 1997, Armstrong et al., 2001, Hashemi-Najafabadi et al., 2006). Additionally, covalently linking of PEG to the RBC surface can mask antigenic sites, thus offering the potential for a universal donor RBC and for improved drug delivery systems (Garratty, 2008, Bradley et al., 2002). Though Pluronic F68 has shown promising results for the treatment of some hyper-viscosity disorders (e.g., sickle cell disease and myocardial infarction) (Armstrong et al., 2004, Orringer et al., 2001), adverse reactions attributed to unsaturated chains of a diblock PPG–PEG component of the copolymer have been observed in some patients (Moghimi et al., 2004).

As an alternative to linear PEG-containing polymers, we propose the use of an amphiphilic star-shaped PEG polymer based on cholic acid core or CA(PEG)4. Star-shaped PEG polymers may show particular promise for improving blood circulation in chronic disorders because the star conformation provides a smaller hydrodynamic radius and lower viscosity than a linear PEG of the same molecular mass (Lin and Zhang, 2010, Lapienis, 2009). Cholic acid, which is a natural bile acid produced in the human liver, is currently used for biomedical and supra-molecular applications (Hofmann, 1995, Tamminen and Kolehmainen, 2001, Virtanen and Kolehmainen, 2004). A series of cholic acid polymer derivatives have been used for drug delivery systems, molecular recognition, dental fillings, and bone repairing materials (Enhsen et al., 1998, Albert and Feigel, 1994, Zhu and Nichifor, 2002). It is now recognized that the incorporation of a bio-compound such as cholic acid into polymers can improve biological compatibility, activity and safety for biomedical applications (Benrebouh et al., 2000, Denike and Zhu, 1994). We thus hypothesize that such amphiphilic star-shaped CA(PEG)4 polymers may be useful for coating RBC before blood transfusion or for intravenous injection in the treatment of elevated RBC aggregation, thus providing rheological benefits similar to Pluronic F68 and PEG. The CA(PEG)4 polymers consist of a hydrophobic core of bile acid and four hydrophilic PEG chains on the periphery located on the concave side of cholic acid.

Although prior studies have described several aspects of RBC adsorption and grafting with polymers having similar structural conformations (i.e., amphiphilic hyperbranched polyglycerol) (Liu et al., 2010, Rossi et al., 2010), they have not investigated their effects on RBC aggregation. In the present study, we examined the influence of star-shaped CA(PEG)4 polymers in an attempt to better understand how these polymers inhibit RBC aggregation and may thus be of value for therapeutic use.

Section snippets

Materials

Star-shaped PEG with a cholane core, abbreviated as CA(PEG)4, were synthesized by a previously reported method (Luo et al., 2009) and were characterized by 1H NMR spectroscopy and MALDI-TOF mass spectrometry. Linear PEGs of 2, 5, 7 and 12 kDa were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), whereas the 22.8 kDa linear PEG was obtained from Polymer Laboratories (Church Stretton, UK).

Methods

The molar masses of the various CA(PEG)4 listed in Table 1 were measured by size exclusion

Synthesis and characterization of CA(PEG)4

The CA(PEG)4 polymers with a molar mass >7 kDa and with narrow polydispersity that were prepared in this study were characterized by SEC–MALLS in water and in PBS. Their weight-average molar masses (Mw) and polydispersity indices (PDI) are given in Table 1, and show lower PDI (<1.22) in water than in PBS (PDI<1.47), especially for higher Mw polymers. Also, Mw values obtained in PBS are 1–1.2 times greater than in water. A series of CA(PEG)4 polymers were synthesized by controlled anionic

Discussion

Elevated RBC aggregation and the associated increase in blood viscosity are recognized risk factors for cardiovascular diseases (Lowe et al., 1997). Although Pluronic F68 (poloxamer 188), which is a PEG–PPG–PEG triblock copolymer, can reduce RBC aggregation, it has been shown to induce adverse reactions in some patients (Moghimi et al., 2004, Szebeni, 2005) that has been attributed to the presence of unsaturation associated with a diblock PEG–PPG component of the copolymer. Synthesis of star

Conclusion

A series of star-shaped CA(PEG)4 polymers with different molar masses and narrow molar mass distributions were synthesized by anionic polymerization. The hydrodynamic radius of these polymers, as determined by intrinsic viscosity measurements, clearly indicated the small and compact structure of star shaped polymers (<2 nm). The polymers also demonstrated shear-thinning behavior in isotonic phosphate buffer (PBS). The extent of inhibition of RBC aggregation by star-shaped CA(PEG)4, as determined

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research (CMI-72323), Heart and Stroke Foundation of Canada (PG-05–0313), and by the National Institutes of Health of USA (RO1 HL078655).

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