Characterization of human erythrocytes as potential carrier for pravastatin: an in vitro study

Abstract

Drug delivery systems including chemical, physical and biological agents that enhance the bioavailability, improve pharmacokinetics and reduce toxicities of the drugs. Carrier erythrocytes are one of the most promising biological drug delivery systems investigated in recent decades. The bioavailability of statin drugs is low due the effects of P-glycoprotein in the gastro-intestinal tract as well as the first-pass metabolism. Therefore in this work we study the effect of time, temperature as well as concentration on the loading of pravastatin in human erythrocytes to be using them as systemic sustained release delivery system for this drug. After the loading process is performed the carriers' erythrocytes were physically and cellulary characterized. Also, the in vitro release of pravastatin from carrier erythrocytes was studied over time interval. Our results revealed that, human erythrocytes have been successfully loaded with pravastatin using endocytosis method either at 25(o)C or at 37(o)C. The loaded amount at 10 mg/ml is 0.32 mg/0.1 ml and 0.69 mg/0.1 ml. Entrapment efficiency is 34% and 94% at 25(o)C and 37(o)C respectively at drug concentration 4 mg/ml. Moreover the percent of cells recovery is 87-93%. Hematological parameters and osmotic fragility behavior of pravastatin loaded erythrocytes were similar that of native erythrocytes. Scanning electron microscopy demonstrated that the pravastatin loaded cells has no change in the morphology. Pravastatin releasing from carrier cell was 83% after 23 hours in phosphate buffer saline and decreased to 72% by treatment of carrier cells with glutaraldehyde. The releasing pattern of the drug from loaded erythrocytes obeyed first order kinetics. It concluded that pravastatin is successfully entrapped into erythrocytes with acceptable loading parameters and moderate morphological changes, this suggesting that erythrocytes can be used as prolonged release for pravastatin.

Keywords: drug delivery; erythrocytes; osmotic fragility; pravastatin.

Figure 1

Effect of pravastatin incubation time and drug concentration on the percent of pravastatin loading on human carrier erythrocytes at 25^oC by endocytosis. The highest loading efficiency obtained when concentration 4 mg/ml is used for incubation time 1 hour. Data is expressed as mean ± SD, Six samples in each group (N = 6).

Figure 2

Effect of pravastatin incubation time and drug concentration on the percent of pravastatin loading on human carrier erythrocytes at 37^oC by endocytosis. The highest loading efficiency obtained when concentration 4mg/ml is used for incubation time 2 hours. Data is expressed as mean ± SD, Six samples in each group (N = 6).

Figure 3

Scanning electron micrograph of pravastatin loaded erythrocytes by endocytosis. A) Control erythrocytes, B) Pravastatin loaded erythrocytes, morphological features like the control one. Magnification is X5000.

Figure 4

Percent of pravastatin and hemoglobin release from loaded erythrocytes in PBS and plasma. Data were tested by one-way analysis of variance and represented as mean ± SD. Three samples in each group (N = 3). Bonferroni multiple comparison tests using SPSS software was performed to determine differences between mean values at (P ≤ 0.01).