Translocating the blood-brain barrier using electrostatics

Abstract

Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components-red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC). To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.

Keywords: blood cells; blood-brain barrier; cell surface charge; drug targeting; zeta-potential.

Figure 1

Zeta-potential of blood components and endothelial cells of mammals in the absence and in the presence of TMA-DPH. (A) RBC (4 × 10⁵ cells/mL), platelets (4 × 10⁶ platelets/mL), and PBMC (4 × 10⁵ cells/mL), HUVEC and BCEC (1 × 10⁵ cells/mL) were incubated with TMA-DPH (54 μM) at 25°C and zeta-potential was measured. Data shown as mean ± SEM; each group value is an average of at least two independent measurements. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs. unlabeled samples, t-test; and ^#P < 0.05, ^##P < 0.01 vs. BCEC, One-Way ANOVA, Bonferroni's multiple comparison test. (B) Schematic representation of TMA-DPH localization in the lipid membrane. The cationic trimethylamino group of TMA-DPH (in blue) locates near the polar heads of phospholipids; anionic phospholipids are represented in red and zwitterionic in yellow.

Figure 2

Relation between zeta-potential and fraction of negatively charged phospholipids in LUVs constituted by POPC and POPG. LUVs (200 μM) were prepared in PBS buffer and zeta-potential measured at 25°C. Each group value is an average of at least two independent measurements; data shown as mean ± SEM. Solid line represents an exponential function fit to the data. It is noteworthy that a change from ζ = −10 mV to ζ = −15 mV (corresponding to a change from RBC/PBMC/platelets to BCEC) is equivalent to a 2-fold increase in surface charge density (inserted arrows).

Figure 3

Zeta-potential of membrane model systems of POPC and POPG. LUVs were incubated with TMA-DPH (54 μM) at 25°C and zeta-potential was measured. Lipid concentration was kept constant at 200 μM. Data shown as mean ± SEM; each group value is an average of at least two independent measurements. ^**P < 0.01 vs. unlabeled samples, t-test; and ^#P < 0.05, ^##P < 0.01 vs. 15% POPG, One-Way ANOVA, Bonferroni's multiple comparison test.

Figure 4

Correlation between the total percentage of negatively charged phospholipids (PS + PI) in the cells membrane and zeta-potential value. For PBMC only PS was accounted for (Table 2). BCEC are a particular case where the high percentage of anionic lipids has a strongly negative zeta-potential value as a counterpart.