(409e) L-Glutamine Delivery to Erythrocytes Via DOPC-DPPG Mixed Liposomes: A Facile System Towards the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is an erythrocyte-based chronic mortal disease resulting from a point mutation in the β chain gene in the hemoglobin A (HbA) molecule. It is known to be one of the most common inherited diseases in the world with a low life expectancy. Although several treatment methods are applied, the pathophysiology of the syndrome is complex and there is still a serious need for development of an effective method for inhibiting or preventing the polymerization and thus sickling of the erythrocytes. Erythrocyte polymerization in SCD is mostly seen as a result of the decrease in the amount of ions and water in the cell, i.e. dehydration and deoxygenation of erythrocytes. Deoxygenated and dehydrated erythrocytes become susceptible to clustering, causing clogging of blood vessels which then leads to crisis. Therefore, development of a treatment method that can effectively prevent deoxygenation of erythrocytes or reduce the oxidative stress of sickle erythrocytes is one of the important issues towards the treatment of SCD. Among a wide variety of potential drug carriers, liposomes are advantageous and preferable with their easy preparation and biocompatibility.

Within this context, in this study we aim to encapsulate L-Glutamine (L-Gln) in liposomes and design a drug carrier system opposed to deoxygenation of erythrocytes in SCD. L-Gln is known to inhibit oxidative stress and its usage is also approved by the US Food and Drug Administration (FDA) to reduce acute pain in SCD patients. To our knowledge, this is the first study that suggests and develops a liposomal drug delivery system for L-Gln administration to erythrocytes and investigates the interactions between red blood cells and drug loaded liposomes. With this scope, the results of this study provide important steps towards obtaining an effective treatment method for SCD.

As a first step of developing drug carriers, we prepared L-Glutamine loaded liposomes composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) & 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG). We have studied the effect of increasing L-Gln content in liposomes by encapsulating L-Gln in three different concentration, namely, 20 mM, 40 mM and 60 mM. In addition, we investigated the physicochemical properties of both unloaded and loaded liposomes as well as analyzing their interactions with red blood cells. Therefore, preliminary results were obtained for further usage of L-Gln encapsulated liposomes towards the reduction and elimination of deoxidation of erythrocytes.

Liposomes were prepared with the well-known thin film hydration method. Afterwards, morphological analysis and physicochemical characterizations of the empty & L-Gln loaded liposomes were performed via several methods such as zeta potential measurements, transmission electron microscopy (TEM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR). Also, drug encapsulation efficiency and release behavior of the liposomes were characterized via high pressure liquid chromatography (HPLC). Effect of the encapsulated amount of L-Gln was investigated by encapsulating the drug at three different concentrations (i.e.20 mM, 40 mM and 60 mM). Investigation of the interactions between drug loaded liposomes and erythrocytes were implemented with hemolysis experiments and also erythrocytes were visualized via optical microscope and SEM before/after incubating red blood cells with drug loaded liposomes.

The sizes of the liposomes were obtained as 179.9±10.4, 182.3±12.1, 185.0±13.3 and 200.2±15.2 nm for unloaded and for 20 mM, 40 mM, 60 mM of L-Gln loaded liposomes, respectively. PDI values of liposomes were determined to be in range of 0.321 to 0.404, which indicate the homogeneous size distribution of liposomes within the suspension. SEM and TEM images (presented in the Figure (a)-(g)) had also supported the spherical liposome formation and also, sizes of the liposomes in both SEM and TEM images were determined to be in good agreement with the results of size measurements obtained with Zeta sizer. Zeta potential values of the liposomes were obtained to be in -22.2±1.3 mV and -20.5±1.6 mV range for both unloaded and drug loaded liposomes, which are in good correlation with the results reported for DOPC/DPPG mixed liposomes in the literature. Thermal analysis with DSC thermograms had showed us that the lipids melt cooperatively in the bilayers and they are fully miscible. Moreover, as encapsulated L-Gln amount is increased, melting temperature of the liposomes displayed a shift to lower temperatures, supporting the incorporation of drug within liposome structure had induced a more fluid bilayer. Also, FTIR results indicated the formation of hydrogen bonds between the head groups of lipids and L-Gln. The encapsulation efficiency of L-Gln was determined to be the higher than the ones reported in the literature: 83.6%, 87.1% and 84.9% for 20 mM, 40 mM and 60 mM Gln, respectively. From in vitro drug release studies, it was found that after 6 hours, release profile had reached to equilibrium and liposomes loaded with 40 mM and 60 mM of L-Gln had released 42.4% and 45.7% of L-Gln, respectively.

After characterizing the physicochemical properties of the liposomes and release profile of L-Gln, the effect of the presence of L-Gln loaded liposomes on the erythrocytes was also investigated through hemolysis assay and also via the images acquired with optical microscopy and SEM. From the hemolysis experiments, after incubation of drug loaded liposomes (with varying L-Gln concentration, i.e. 20 mM, 40 mM and 60 mM) with erythrocytes at 37 ºC for 3 hours, we determined that the presence of liposomes and their interactions with erythrocytes do not cause any significant damage on the structure and morphology of the cells, as this result was also supported with the optical microscopy images of the cells before/after the incubation. Moreover, SEM images of the erythrocytes were acquired and again as supported with the optical microscopy images, it was concluded that the presence of L-Gln loaded liposomes had not only caused no hemolysis, but also, they provide a synergistic effect on the preservation of the structure of red blood cells. We propose that adsorption of liposomes to the membranes of erythrocytes had increased the stability of the cells and the presence of lipids support the erythrocyte membrane to retain its integrity (Figure (h) and (i)). In fact, this is another important result of the study which proves the positive effect of the presence of L-Gln loaded liposomes on erythrocytes.

In conclusion, L-Gln loaded PC/PG liposomes provide preliminary yet promising results in terms of developing a new drug delivery platform towards SCD, for the first time in the literature. Although in vivo experiments should also be conducted in the future studies, we believe PC/PG liposomal carriers developed in this study carry a great potential to be used in prevention of deoxidation of erythrocytes which induce polymerization and sickling of cells and lead to painful crisis in SCD.