Drug Exchange between Albumin Nanoparticles and Erythrocyte Membranes

Abstract

The effects of thioridazine (TDZ) and chlorpromazine (CPZ) and bovine serum albumin nanoparticles (BSA-NPs) on erythrocyte membranes have been investigated. Two kinds of hemolytic assays were used; hemolysis under hypotonic conditions and hemolysis in physiological conditions. Under hypotonic conditions for 50% hemolysis, both TDZ and CPZ have a biphasic effect on membranes; namely, stabilization at low concentrations and destabilization after reaching a critical concentration. In physiological conditions, there are other critical concentrations above which both drugs hemolyse the erythrocites. In each case, the critical concentrations of TDZ are lower than those of CPZ, which is consistent with the ratio of their partition coefficients. When BSA-NPs are added to the erythrocyte suspension simultaneously with the drugs, the critical concentrations increase for both drugs. The effect is due to the incorporation of a portion of drug substances into the BSA-nanoparticles, which consequently leads to the decrease of the active drug concentrations in the erythrocyte suspension medium. Similar values of the critical concentrations are found when the BSA-NPs are loaded with the drugs before their addition to the erythrocyte suspension in which case the events of the partition are: desorption of the drug from BSA-NPs, diffusion through the medium, and adsorption on erythrocyte membranes. This result suggests that the drugs are not influenced by the processes of adsorption and desorption onto and out of the BSA-NPs, and that the use of BSA-NPs as drug transporters would allow intravenous administration of higher doses of the drug without the risk of erythrocyte hemolysis.

Keywords: bovine serum albumin nanoparticles; erythrocyte membranes; hemolytic assay; phenothiazine drugs.

Figure 1

Shematic presentation of the hemolytic assays performed with simultaneously added drugs and BSA-NPs (A) and with drug-loaded BSA-NPs (B).

Figure 2

Release profiles of TDZ and CPZ from loaded BSA-NPs, presented as percentage of the initial amount of loaded drug.

Figure 3

Influence of the drugs and combined influence of drugs and BSA-NPs on hypotonic hemolysis of erythrocytes (hematocrit 0.95%, 66 mM NaCl and 5 mM phosphate buffer, pH 7.4). Incubation time 30 min. The results are averaged over three measurements for each concentration of the respective drug substance. (a) TDZ (solid curve) and TDZ plus 0.32 mg/mL BSA-NPs (dashed curve). (b) CPZ (solid curve) and CPZ plus 0.32 mg/mL BSA-NPs (dashed curve).

Figure 4

Relative hemolysis (R.H.) caused by the action of TDZ and CPZ on erythrocytes at physiological conditions (hematocrit 0.95%, 149 mM NaCl, 5 mM phosphate buffer, pH 7.4). Incubation time 30 min. The results are averaged over three measurements for each concentration of the respective drug substance.

Figure 5

Influence of the drugs and combined influence of drugs and BSA-NPs on the hemolysis of erythrocytes in physiological conditions (hematocrit 0.95%, 149 mM NaCl and 5 mM sodium phosphate, pH 7.4). Incubation time 30 min. (a) TDZ alone (circles, solid blue curve), TDZ plus 0.32 mg/mL BSA-NPs (squares, dashed orange curve), 0.32 mg/mL BSA-NPs loaded with TDZ (triangles, solid green curve. (b) CPZ alone (circles, solid red curve); CPZ plus 0.32 mg/mL BSA-NPs (squares, dashed purple curve), 0.32 mg/mL BSA-NPs loaded with CPZ (triangles, solid green curve). The results are averaged over three measurements for each concentration of the respective drug substance.

Figure 6

Absorption spectra of supernatants of erythrocytes suspensions with different amount of TDZ (a–d) and CPZ (e–h) without BSA-NPs or with 0.32 mg/mL BSA-NPs: Equal amount of TDZ (a) and of CPZ (e), provoking hemolysis without BSA-NPs and not provoking hemolysis in the presence of BSA-NPs. Different amount of TDZ (b) and CPZ (f) without and with BSA-NPs, both below the threshold concentration $C_{\mathrm{sat}}$ for the onset of hemolysis. Different amount of TDZ (c) and CPZ (g) without and with BSA-NPs, both above $C_{\mathrm{sat}}$ and below $C_{\mathrm{sol}}$. Different amount of TDZ (d) and CPZ (h) without and with BSA-NPs, both above $C_{\mathrm{sol}}$ for complete hemolysis.