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

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.

Figure 1

Structure of the star-shaped PEG with a cholane core CA(PEG)₄. Methylene protons of PEG were present at about 3.4 – 3.5 ppm.

Figure 2

The intrinsic viscosity [η] (circles) and hydrodynamic radius (R_h) (squares) against the number-average molar mass (M_w) for a series of solutions of CA(PEG)₄ polymers in water at 25 °C. CA(PEG)₄ polymers had a compact structure with R_h values less than about 2 nm and small [η] values below about 2 mL/g.

Figure 3

Logarithmic plot of the intrinsic viscosity as a function of the molar mass of CA(PEG)₄ polymers in double distilled water at 25°C. The solid line shows the fit to the Mark Houwink equation, where K = −5.445 and a = 1.3 with adjusted R² = 0.985.

Figure 4

Semi-logarithmic plot of the dependence the flow curves on the molar mass of the star-shaped CA(PEG)₄ polymers at 25 °C in (A) phosphate buffer saline solution (PBS) at 0.3% (w/v) and (B) in water. Flow viscosity curves show that CA(PEG)₄ in PBS exhibits pronounced shear thinning behavior, especially at high molar mass fractions, whereas shear thinning is less pronounced in water.

Figure 5

Viscosity ratio (apparent viscosity at 0.15 s⁻¹ divided by that at 94 s⁻¹) of human RBC suspended in autologous plasma at 25°C with CA(PEG)₄ solution (6.7 mg/mL) normalized to control (0 mg/mL) and presented as mean ± SD. Measurements were in duplicates for each donor (n = 3). Compared to unity corresponding to the polymer-free suspension (control), inhibition of RBC aggregation was close to significance (p = 0.08).

Figure 6

Aggregation of RBC suspended in autologous plasma measured using a Myrenne aggregometer. Aggregation indices are M at stasis (A) and M1 at 3 s⁻¹ (B) normalized to control (i.e., buffer added without polymer) and presented as mean ± SD for CA(PEG)₄ at different concentrations (1.3, 4.0 and 6.7 mg/mL) and linear PEG at a concentration of 6.7 mg/mL. Values for M and M1 < 1 indicate inhibition of RBC aggregation. Measurements were in duplicates for each donor (n = 3). Results demonstrate that at concentrations of 4.0 and 6.7 mg/mL, star-shaped CA(PEG)₄ are able to reduce RBC aggregation (p < 0.01).

Figure 7

Photomicrograph images of RBC in plasma at 40x magnification. The CA(PEG)₄ sample was obtained by adding the polymer at a concentration of 6.7 mg/mL to RBC and plasma. Normal discocytic RBC morphology is seen in both samples.