Development of a liposomal nanodelivery system for nevirapine
© Ramana et al; licensee BioMed Central Ltd. 2010
Received: 10 April 2010
Accepted: 13 July 2010
Published: 13 July 2010
The treatment of AIDS remains a serious challenge owing to high genetic variation of Human Immunodeficiency Virus type 1 (HIV-1). The use of different antiretroviral drugs (ARV) is significantly limited by severe side-effects that further compromise the quality of life of the AIDS patient. In the present study, we have evaluated a liposome system for the delivery of nevirapine, a hydrophobic non-nucleoside reverse transcriptase inhibitor. Liposomes were prepared from egg phospholipids using thin film hydration. The parameters of the process were optimized to obtain spherical liposomes below 200 nm with a narrow polydispersity. The encapsulation efficiency of the liposomes was optimized at different ratios of egg phospholipid to cholesterol as well as drug to total lipid. The data demonstrate that encapsulation efficiency of 78.14% and 76.25% were obtained at egg phospholipid to cholesterol ratio of 9:1 and drug to lipid ratio of 1:5, respectively. We further observed that the size of the liposomes and the encapsulation efficiency of the drug increased concomitantly with the increasing ratio of drug and lipid and that maximum stability was observed at the physiological pH. Thermal analysis of the drug encapsulated liposomes indicated the formation of a homogenous drug-lipid system. The magnitude of drug release from the liposomes was examined under different experimental conditions including in phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum or in the presence of an external stimulus such as low frequency ultrasound. Within the first 20 minutes 40, 60 and 100% of the drug was released when placed in PBS, DMEM or when ultrasound was applied, respectively. We propose that nevirapine-loaded liposomal formulations reported here could improve targeted delivery of the anti-retroviral drugs to select compartments and cells and alleviate systemic toxic side effects as a consequence.
According to the World Health Organization, more than 40 million people have been presently infected with Human Immunodeficiency Virus type 1 (HIV-1) globally. Highly Active Antiretroviral Therapy (HAART), which consists of a combination of a minimum of three antiretroviral (ARV) drugs, is the primary treatment currently available for efficient management of AIDS [1, 2]. The various types of ARVs that are used in HAART could be categorized into nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors (nRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), viral fusion inhibitors, integrase inhibitors, maturation inhibitors and fixed dose combination . These drugs have a potential to manage the chronic infection but not to treat the disease . The bioavailability of many of the ARV drugs is considerably low and erratic due to the substantial first pass metabolism and degradation in the gastrointestinal tract. Given the short half-life of the drugs, frequent administration of the drugs is required at relatively higher doses, often leading to low patient compliance . If adherence falls below 95% level, the therapeutic effectiveness is reduced below 50% . Immunologically privileged compartments of the body including the central nervous system, lymphatic system and the macrophages are characteristically inaccessible to a majority of the ARV drugs thus serving as viral reservoirs . The inability to maintain therapeutic concentration of the drugs for longer durations significantly contributes to multidrug-resistance . Furthermore, the prolonged use of ARVs frequently leads to toxic side effects resulting in the deterioration in the quality of life and incompliance to therapy . Nevirapine is a hydrophobic NNRTI that non-competitively binds to an allosteric non-substrate binding site of the reverse transcriptase (RT) [8, 9]. Nevirapine, the first ARV member of non-nucleoside reverse transcriptase inhibitor approved by the Food and Drug Administration (FDA) for HIV and an important component of HAART, is typically the primary choice for efficient viral suppression. Unfortunately, the use of nevirapine is frequently accompanied by severe side-effects that include CNS toxicity, hepatotoxicity, insomnia, confusion, memory loss, depression, rash, nausea, dizziness, Stevens-Johnson syndrome, toxic epidermal necrolysis and hyperlipidemia [8–16]. It has also been reported to cause severe liver toxicity within first six weeks of treatment. The US FDA has issued a 'black box label' on nevirapine due to its hepatotoxicity . The use of nevirapine has been restricted except in cases where the benefit to the patient exceeds the risk. Therefore the development of a delivery system for sustained and targeted release of nevirapine can enhance the clinical potential of this antiretroviral drug. Nevirapine also reduces the level of certain co-administered drugs including the antiretroviral drugs indinavir, lopinavir, efavirenz. In order to prevent such undesired interactions, encapsulation of nevirapine in a carrier is expected to be beneficial. Given the paradoxical context, there exists a need to develop targeted and sustained drug delivery systems to reduce the frequency of dose administration on the one hand and to maintain therapeutic concentration of the drug for extended periods with enhanced efficacy on the other hand which could also improve patient compliance. The liposomal carrier system is expected to reduce the side-effects due to sustained release of the drug and provide sufficient cellular uptake due to its nano-dimensions.
A range of novel strategies are currently being developed for efficient delivery of ARV drugs. Efficient delivery could be achieved by encapsulating the drug or by attaching it with a carrier system [17–19]. Several delivery systems have been reported for the delivery of ARV drugs including bioadhesive coated matrix tablets [20, 21], ceramic implants , liposomes [23–26], solid colloidal nanoparticles [27–30], dendrimers , micelles & microemulsion , nanopowders  and suspensions . Liposomes are nanocarriers that range from 25 nm to several microns and are prepared using combinations of natural or synthetic phospholipids and cholesterol . Liposomes incorporate hydrophilic drugs through an aqueous core or entrap hydrophobic drugs using phospholipid bilayer(s) which surrounds the aqueous core. Since some of the cells of the immune system like the macrophages and microglial cells could serve as the viral reservoirs, liposomes could potentially target ARV drugs into the infected cells thereby improving the efficacy and reducing the side-effects . The primary aim of the present study was to develop and characterize nevirapine-loaded liposomes and to investigate the effect of various parameters on the size and the encapsulation efficiency of the liposomes including the lipid composition, drug-lipid ratio and pH of the medium. The release kinetics of nevirapine in solutions at varying pH and culture medium in the presence and absence of an external stimulus were determined.
Materials and methods
Methanol, phosphotungstic acid, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sucrose, chloroform, hydrochloric acid were purchased from Merck Chemicals, India and used as such without further purification. Egg phosphatidyl choline (EPC) was procured from Sigma-Aldrich, USA. Nevirapine was a kind gift from Bohringer Ingelheim, Germany.
Preparation of Liposomes
Egg phospholipids were extracted from yellow yolk by the modified Singleton-Gray method . The lipid composition of egg phospholipids have been identified using the GC-MS (Agilent technologies, Model 7890 A series, GC with 5975C Mass spectrometer). The results indicate that the egg phospholipid contains three different lipid constituents such as PLPC (89%), POPE (3%) and cholesterol (6%). Liposomes were prepared using the thin film hydration technique. Briefly, 100 mg/mL of phospholipids in chloroform taken in a clean moisture-free container was purged with nitrogen gas to remove the solvent. Five mL of phosphate buffered saline (PBS), pH 7.4, were added to the container and the mixture was warmed at 60°C for 30 minutes. The solution was then extruded through polycarbonate membranes of 200 nm pore size using an extruder (Liposofast Basic, Avestin, Canada) for ten cycles to obtain extruded liposomes. The liposomes were lyophilized (Virtis Model Benchtop K, USA) and stored at -20 °C in air-tight vials.
Nevirapine loaded liposomes were prepared dissolving eight different ratios of drug to phospholipids (1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:10). Briefly, a total amount of lipid consisting of 10 mg of phospholipids in chloroform and different quantities of nevirapine was dissolved in chloroform and the liposomes were prepared as explained above.
Particle Size Analysis
The particle size of the liposomes and drug loaded liposomes were determined using laser diffraction method (Microtrac Blue wave, Japan) at room temperature. Five mL of the sample was introduced into the particle size analyzer at 50% flow rate to measure the mean size and size distribution of liposomes and drug loaded liposomes.
Two mg of liposome samples were loaded in aluminum pans along with the standard reference aluminum in the differential scanning calorimeter (Q20, TA Instruments, USA). The DSC was recorded between 10°C and 90°C at a scan rate of 10°C/min for three cycles.
Determination of Encapsulation Efficiency
The extruded liposomal samples were centrifuged at 3,000 rpm (Eppendorf 3340R, Germany) at 4°C to pelletize the unencapsulated drug. The supernatant was centrifuged at 10,000 rpm to pelletize the drug loaded liposomes . The pellet was then treated with 1% Triton X-100 (Sigma-Aldrich, USA) to disrupt the liposomes. The sample was centrifuged at 3000 rpm again to pelletize the drug alone. The supernatant was removed and the pellet was resuspended and the concentration of the encapsulated drug was measured as absorbance at 284 nm using UV-visible spectrophotometer (Lambda 25, Perkin Elmer, USA). The absorbance was converted into drug concentration using a standard curve.
All experiments were carried out in triplicate.
Dialysis bags (Dialysis membrane 110, Hi Media, India) were immersed in water for one hour to remove the preservatives followed by rinsing in phosphate buffered saline (PBS) solution. The drug encapsulated liposomes were placed in PBS and loaded in the dialysis bag. The bag was sealed at both the ends and immersed in 4 mL of PBS with 10% methanol . The release of the drug was evaluated at three different pH values (1.2, 7.4 and 9.0). A pH of 1.2 was maintained using 0.1 M HCl -KCl buffer while pH 9.0 was maintained using 0.1 M phosphate buffer. In order to evaluate the influence of proteins on the release of nevirapine from the liposomes, Dulbecco's Modified Eagle's Medium (DMEM, HiMedia, India) supplemented with 10% fetal bovine serum (FBS, HiMedia, India) was used as the release medium. The effect of ultrasound on the release profile of the drug from the liposomes was studied in PBS (pH 7.4) as the release medium. Low frequency ultrasound (20 KHz) was applied using a bath sonicator (UT 002, ABM, India) for the entire duration of the release study. For all drug release studies, 4 ml of the release medium was withdrawn for analysis at different time intervals (0-25 hours) and replaced with 4 mL of fresh medium. The amount of drug released was measured as absorbance using a UV-visible spectrophotometer (Lambda 25, Perkin Elmer, USA). The absorbance was converted into percentage release using a standard curve.
Analysis of Variance (Two-way Anova) was performed to determine the statistical significance (p < 0.05) for percentage encapsulation (n = 3) and percentage of drug release (n = 3) under various experimental conditions. If statistically significant, a post-hoc Tukey test was performed to determine which means were different from the others.
Results & Discussion
After the initial aggregation, two distinct size groups of liposomes - nano and micro size liposomes were formed at both acidic (Figure 2G) and alkaline pH conditions (Figure 2I). As a function of time the size of the particles decreased and larger fraction of the liposomes were seen in the nanoscale range at both acidic (Figure 2J) and basic (Figure 2L) pH conditions.
Liposomes of uniform diameters were prepared using thin film hydration and extrusion technique and a hydrophobic non-nucleoside reverse transcriptase inhibitor, nevirapine, was successfully encapsulated in the liposomes. The best encapsulation was observed at an egg phosholipid to cholesterol ratio of 9:1 which also showed a prolonged release of nevirapine up to 1320 minutes at physiological pH. Presence of proteins in the medium and external stimuli like low frequency ultrasound was found to enhance the rate of drug release. The use of ultrasound leading to higher magnitude of drug release thus points to a potentially novel approach towards anti-retroviral therapy. Presence of cholesterol in the liposomes offers stability against fluidizing action of proteins without preventing the disruption of the liposomal architecture by ultrasound.
The authors wish to record their gratitude to Nano Mission Council, Department of Science and Technology and Prof. T.R. Rajagopalan R&D Grant, SASTRA University, for financial support.
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