Pharmaceutical guide

Tablet: Manufacturing methods/Granulation

Introduction

Granulation may be defined as a size enlargement process which converts small particles into physically stronger & larger agglomerates.Granulation method can be broadly classified into two types: Wet granulation and Dry granulation

Ideal characteristics of granulesThe ideal characteristics of granules include spherical shape, smaller particle size distribution with sufficient fines to fill void spaces between granules, adequate moisture (between 1-2%), good flow, good compressibility and sufficient hardness.The effectiveness of granulation depends on the following propertiesi) Particle size of the drug and excipientsii) Type of binder (strong or weak)iii) Volume of binder (less or more)iv) Wet massing time ( less or more)v) Amount of shear appliedvi) Drying rate ( Hydrate formation and polymorphism)

Wet granulation

The most widely used process of agglomeration in pharmaceutical industry is wet granulation. Wet granulation process simply involves wet massing of the powder blend with a granulating liquid, wet sizing and drying.

Important steps involved in the wet granulation

i) Mixing of the drug(s) and excipientsii) Preparation of binder solutioniii) Mixing of binder solution with powder mixture to form wet mass. screens).iv) Coarse screening of wet mass using a suitable sieve (6-12).v) Drying of moist granules. screen).vi) Screening of dry granules through a suitable sieve (14 20vii) Mixing of screened granules with disintegrant, glidant, and lubricant.

Limitation of wet granulation

i)The greatest disadvantage of wet granulation is its cost. It is an expensive process because of labor, time, equipment, energy and space requirements.ii)Loss of material during various stages of processingiii)Stability may be major concern for moisture sensitive or thermo labile drugsiv)Multiple processing steps add complexity and make validation and control difficultv)An inherent limitation of wet granulation is that any incompatibility between formulation components is aggravated.

Special wet granulation techniques

i) High shear mixture granulationii) Fluid bed granulationiii) Extrusion-spheronizationiv) Spray drying

High shear mixture granulation

High shear mixture has been widely used in Pharmaceutical industries for blending and granulation. Blending and wet massing is accompanied by high mechanical agitation by an impeller and a chopper. Mixing, densification and agglomeration are achieved through shear and compaction force exerted by the impeller.Advantages:i) Short processing timeii) Less amount of liquid binders required compared with fluid bed.iii) Highly cohesive material can be granulated.

Fluid bed granulation

Fluidization is the operation by which fine solids are transformed into a fluid like state through contact with a gas. At certain gas velocity the fluid will support the particles giving them free mobility without entrapment.Fluid bed granulation is a process by which granules are produced in a single equipment by spraying a binder solution onto a fluidized powder bed. The material processed by fluid bed granulation are finer, free flowing and homogeneous.

Extrusion and Spheronization

It is a multiple step process capable of making uniform sized spherical particles. It is primarily used as a method to produce multi-particulates for controlled release application.Advantages:

i) Ability to incorporate higher levels of active components without producing excessively larger particles.ii) Applicable to both immediate and controlled release dosage form.

Spray drying granulation

It is a unique granulation technique that directly converts liquids into dry powder in a single step. This method removes moisture instantly and converts pumpable liquids into a dry powder.Advantages:i) Rapid processii) Ability to be operated continuouslyiii) Suitable for heat sensitive product

Lists of equipments for wet granulation

High Shear granulation:i)Little ford Lodgie granulatorii)Little ford MGT granulatoriii)Diosna granulatoriv)Gral mixer

Granulator with drying facility:i) Fluidized bed granulatorii) Day nauta mixer processoriii) Double cone or twin shell processoriv) Topo granulatorSpecial granulator:i) Roto granulatorii) Marumerizer

Current topics related to wet granulation

I.Hydrate formationFor example, theophylline anhydrous during high shear wet granulation transfers to theophylline monohydrate. The midpoint conversion occurs in three minutes after the binder solution is added.For online monitoring of the transformation from one form to another, Raman spectroscopy is most widely used.II.Polymorphic transformationThe drying phase of wet granulation plays a vital role for conversion of one form to another.

For example, glycine which exist in three polymorphs that is α β γ . γ is the most stable form and αis the metastable form. The stable Glycine polymorph (γ) converts to metastable form (α) when wet granulated with microcrystalline cellulose.

Dry granulation

Introduction

In dry granulation process the powder mixture is compressed without the use of heat and solvent. It is the least desirable of all methods of granulation. The two basic procedures are to form a compact of material by compression and then to mill the compact to obtain a granules. Two methods are used for dry granulation. The more widely used method is slugging, where the powder is precompressed and the resulting tablet or slug are milled to yield the granules. The other method is to precompress the powder with pressure rolls using a machine such as Chilosonator.

Advantages

The main advantages of dry granulation or slugging are that it uses less equipments and space. It eliminates the need for binder solution, heavy mixing equipment and the costly and time consuming drying step required for wet granulation. Slugging can be used for advantages in the following situations:i) For moisture sensitive materialii) For heat sensitive materialiii) For improved disintegration since powder particles are not bonded together by a binder

Disadvantages

i) It requires a specialized heavy duty tablet press to form slugii) It does not permit uniform colour distribution as can beiii) Achieved with wet granulation where the dye can be incorporated into binder liquid.iv) The process tends to create more dust than wet granulation, increasing the potential contamination.

Steps in dry granulation

i) Milling of drugs and excipientsii) Mixing of milled powdersiii) Compression into large, hard tablets to make slugiv) Screening of slugsv) Mixing with lubricant and disintegrating agentvi) Tablet compression

Two main dry granulation processes

Slugging process

Granulation by slugging is the process of compressing dry powder of tablet formulation with tablet press having die cavity large enough in diameter to fill quickly. The accuracy or condition of slug is not too important. Only sufficient pressure to compact the powder into uniform slugs should be used. Once slugs are produced they are reduced to appropriate granule size for final compression by screening and milling.

Factors which determine how well a material may slugi) Compressibility or cohesiveness of the materii) Compression ratio of powderiii) Density of the powderiv) Machine typev) Punch and die sizevi) Slug thicknessvii) Speed of compressionviii) Pressure used to produce slug

Roller compaction

The compaction of powder by means of pressure roll can also be accomplished by a machine called chilsonator. Unlike tablet machine, the chilsonator turns out a compacted mass in a steady continuous flow. The powder is fed down between the rollers from the hopper which contains a spiral auger to feed the powder into the compaction zone. Like slugs, the aggregates are screened or milled for production into granules.

Formulation for dry granulation

The excipients used for dry granulation are basically same as that of wet granulation or that of direct compression. With dry granulation it is often possible to compact the active ingredient with a minor addition of lubricant and disintegrating agent. Fillers that are used in dry

granulation include the following examples: Lactose, dextrose, sucrose, MCC, calcium sulphate, Sta-Rx® etc.Examples of some tablet formulation prepared by dry granulation

Advancement in Granulations

Steam Granulation

It is modification of wet granulation. Here steam is used as a binder instead of water. Its several benefits includes higher distribution uniformity, higher diffusion rate into powders, more favourable thermal balance during drying step, steam granules are more spherical, have large surface area hence increased dissolution rate of the drug from granules, processing time is shorter therefore more number of tablets are produced per batch, compared to the use of organic solvent water vapour is environmentally friendly, no health hazards to operators, no restriction by ICH on traces left in the granules, freshly distilled steam is sterile and therefore the total count can be kept under control, lowers dissolution rate so can be used for preparation of taste masked granules without modifying availability of the drug. But the limitation is that it is unsuitable for thermolabile drugs. Moreover special equipments are required and are unsuitable for binders that cannot be later activated by contact with water vapour.

Melt Granulation / Thermoplastic Granulation

(24)Here granulation is achieved by the addition of meltable binder. That is binder is in solid state at room temperature but melts in the temperature range of 50 – 80˚C. Melted binder then acts like a binding liquid. There is no need of drying phase since dried granules are obtained by cooling it to room temperature. Moreover, amount of liquid binder can be controlled precisely and the production and equipment costs are reduced. It is useful for granulating water sensitive material and producing SR granulation or solid dispersion. But this method is not suitable for thermolabile substances. When water soluble binders are needed, Polyethylene Glycol (PEG) is used as melting binders. When water insoluble binders are needed, Stearic acid, cetyl or stearyl alcohol, various waxes and mono-, di-, & triglycerides are used as melting binders.

Moisture Activated Dry Granulation (MADG)

(58)It involves moisture distribution and agglomeration. Tablets prepared using MADG method has better content uniformity. This method utilizes very little granulating fluid. It decreases drying time and produces granules with excellent flowability.

Moist Granulation Technique (MGT)

(59)A small amount granulating fluid is added to activate dry binder and to facilitate agglomeration. Then a moisture absorbing material like Microcrystalline Cellulose (MCC) is added to absorb any excess moisture. By adding MCC in this way drying step is not necessary. It is applicable for developing a controlled release formulation.

Thermal Adhesion Granulation Process (TAGP)

(60)It is applicable for preparing direct tableting formulations. TAGP is performed under low moisture content or low content of pharmaceutically acceptable solvent by subjecting a mixture containing excipients to heating at a temperature in the range from about 30ºC to about 130ºC in a closed system under mixing by tumble rotation until the formation of granules. This method utilizes less water or solvent than traditional wet granulation method. It provides granules with good flow properties and binding capacity to form tablets of low friability, adequate hardness and have a high uptake capacity for active substances whose tableting is poor.

Foam Granulation

(61)Here liquid binders are added as aqueous foam. It has several benefits over spray(wet) granulation such as it requires less binder than Spray Granulation, requires less water to wet granulate, rate of addition of foam is greater than rate of addition of sprayed liquids, no detrimental effects on granulate, tablet, or invitro drug dissolution properties, no plugging problems since use of spray nozzles is eliminated, no overwetting, useful for granulating water sensitive formulations, reduces drying time, uniform distribution of binder throughout the powder bed, reduce manufacturing time, less binder required for Immediate Release (IR) and Controlled Release (CR) formulations

Friability Test

“FRIABILITY is the phenomenon where the surface of the tablet is damage or shown a site of damage due to mechanical shock.”

PURPOSE: To evaluate the ability of the tablets to withstand the breakage during the transportation and handling.

APPARATUS:“ROCHE FRIABALITOR”

Roche friabalitor consist of a drum which have an internal diameter between 283 and 291 mm and a depth between 36 and 40 mm, of transparent synthetic polymer with polished internal surfaces, and subject to minimum static build-up (see figure for a typical apparatus). One side of the drum is removable. The tablets are tumbled at each turn of the drum by a curved projection with an inside radius between 75.5 and 85.5 mm that extends from the middle of the drum to the outer wall. The outer diameter of the central ring is between 24.5 and 25.5 mm. The drum is attached to the horizontal axis of a device that rotates at 25 ±1 rpm. Thus, at each turn the tablets roll or slide and fall onto the drum wall or onto each other.

CRITERA:

For tablets with a unit weight equal to or less than 650 mg, take a sample of whole tablets corresponding as near as possible to 6.5 g. For tablets with a unit weight of more than 650 mg, take a sample of 10 whole tablets.

PROCEDURE:

The tablets should be carefully dedusted prior to testing. Accurately weigh the tablet sample, and place the tablets in the drum. Rotate the drum 100 times, and remove the tablets. Remove any loose dust from the tablets as before, and accurately weigh.

Generally, the test is run once. If obviously cracked, cleaved, or broken tablets are present in the tablet sample after tumbling, the sample fails the test. If the results are difficult to interpret or if the weight loss is greater than the targeted value, the test should be repeated twice and the mean of the three tests determined. A maximum mean weight loss from the three samples of not more than 1.0% is considered acceptable for most products.

Effervescent tablets and chewable tablets may have different specifications as far as friability is concerned. In the case of hygroscopic tablets, an appropriate humidity-controlled environment is required for testing.

PRECENTAGE OF FRIABILITY of the tablets of a badge can be find by the following formula:

Percentage Friability = W1 – W2/W1 × 100

Where, W1 = weight of tablets before testing

W2 = weight of tablets after testing.

LIMIT:

According to B.P = Percentage of friability should be not more than 0.8%.

According to U.S.P = Percentage of friability should be not more than 4%

Hardness Test

Hardness is also so called crushing strength. It is the load required to crush the tablet when placed on its edge. There are a variety of presentations for tablets as delivery systems for pharmaceutical agents, such as rapidly disintegrating, slowly disintegrating, eroding, chewable, and lozenge. Each of these presentations places a certain demand on the bonding, structure, and integrity of the compressed matrix. Tablets must be able to withstand the rigors of handling and

transportation experienced in the manufacturing plant, in the drug distribution system, and in the field at the hands of the end users (patients/consumers). Manufacturing processes such as coating, packaging, and printing can involve considerable stresses, which the tablets must be able to withstand. For these reasons, the mechanical strength of tablets is of considerable importance and is routinely measured. Tablet strength serves both as a criterion by which to guide product development and as a quality control specification.

Measure of the mechanical integrity of tablets is their breaking force, which is the force required to cause them to fail (i.e., break) in a specific plane. The tablets are generally placed between two platens, one of which moves to apply sufficient force to the tablet to cause fracture. For conventional, round (circular cross-section) tablets, loading occurs across their diameter (sometimes referred to as diametral loading), and fracture occurs in that plane.

The breaking force of tablets is commonly called hardness in the pharmaceutical literature (“hardness is defined as the resistance of the tablet against the applied force till it breaks”); however, the use of this term is misleading. In material science, the term hardness refers to the resistance of a surface to penetration or indentation by a small probe. The term crushing strength is also frequently used to describe the resistance of tablets to the application of a compressive load. Although this term describes the true nature of the test more accurately than does hardness, it implies that tablets are actually crushed during the test, which often is not the case. Moreover, the term strength in this application can be questioned, because in the physical sciences that term is often used to describe a stress (e.g., tensile strength). Thus, the term breaking force is preferred.

Why do we measure hardness?To determine the need for pressure adjustments on the tableting machine.Hardness can affect the disintegration. So if the tablet is too hard, it may not disintegrate in the required period of time. And if the tablet is too soft, it will not withstand the handling during subsequent.processing such as coating or packaging.In general, if the tablet hardness is too high, we first check its disintegration before rejecting the batch . And if the disintegration is within limit, we accept the batch.If H. is high + disintegration is within time è accept the batch.

APPARATUS FOR MEASURING HARDNESS

1. Monsanto Hardness Tester or Stokes Hardness tester (1930).

2. Pfizer Hardness Tester (1950).

3. Strong cob Hardness Tester.

4. Heberlain Hardness Tester or Schleeniger Hardness tester.

Early measuring devices were typically hand operated. For example, the Monsanto (or Stokes) hardness tester was based on compressing tablets between two jaws via a spring gauge and screw. In the Pfizer hardness tester, the vertically mounted tablet was squeezed in a device that resembled a pair of pliers. In the Strong Cobb hardness tester, the breaking load was applied through the action of a small hydraulic pump that was first operated manually but was later motorized. Problems associated with these devices were related to operator variability in rates of loading and difficulties in proper setup and calibration. Modern testers employ mechanical drives, strain gauge–based load cells for force measurements, and electronic signal processing, and therefore are preferred.

However, the result of Strong cob hardness tester is 1.6 times accurate than the Heberlain hardness tester and 1.4 times more accurate than the Pfizer hardness tester.

Most commonly used apparatus for hardness is electronically operated hardness tester i.e. HEBERLAIN HARDNESS TESTER.

HEBERLAIN HARDNESS TESTER:

This apparatus usually have two platens between which tablets is placed and compressed and the value of the hardness is measured. The platens should be parallel. Their faces should be polished smooth and precision-ground perpendicularly to the direction of movement. Perpendicularity must be preserved during platen movement, and the mechanism should be free of any bending or torsion displacements as the load is applied. The contact faces must be larger than the area of contact with the tablet.

Either the rate of platen movement or the rate at which the compressive force is applied (i.e., the loading rate) should be constant. Maintaining a constant loading rate avoids the rapid buildup of compressive loads, which may lead to uncontrolled crushing or shear failure and greater variability in the measured breaking force. However, constant loading rate measurements may be too slow for real time monitoring of tablet production.

UNIT of measuring hardness is kg/cm2.

CRITERA: Tablet hardness should lies between 5 to 10 kg/cm2.

RESULT LIMIT: ± 5%.

Thickness and diameter of the tablets can also be checked by this Heberlain hardness tester. The mode of the apparatus is set according to the test (hardness, thickness or diameter

Factors Affecting the Hardness:Compression of the tablet and compressive force. Amount of binder. (More binder à more hardness) Method of granulation in preparing the tablet (wet method gives more hardness than direct method, Slugging method gives the best hardness).

Limits:5 kilograms minimum and 8 kilograms maximum.Make hardness test on 5 tablets and then take the average hardness

Bulk Density

Bulk density is a property of powders, granules and other "divided" solids, especially used in reference to mineral components (soil, gravel), chemical substances, (pharmaceutical) ingredients, foodstuff or any other masses of corpuscular or particulate matter. It is defined as the mass of many particles of the material divided by the total volume they occupy. The total volume includes particle volume, inter-particle void volume and internal pore volume.Bulk density is not an intrinsic property of a material; it can change depending on how the material is handled. For example, a powder poured in to a cylinder will have a particular bulk density; if the cylinder is disturbed, the powder particles will move and usually settle closer together, resulting in a higher bulk density. For this reason, the bulk density of powders is usually reported both as "freely settled" (or "poured" density) and "tapped" density (where the tapped density refers to the bulk density of the powder after a specified compaction process, usually involving vibration of the container.)

Weight Variation

Weight variation of individual tablets, the average tablet weight was determined by weighing 20 units or tablets individually using an analytical balance.

Preparation, characterization, and in vitro release study of albendazole-encapsulated nanosize liposomes

Introduction

Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment that are generated in vitro. In 1965, Bangham and colleagues first used a liposomal structure as a model to study the effect of narcotics on lipid bilayer membranes.1 These are useful in terms of biocompatibility, biodegradability, and low toxicity, and can control biodistribution by changing the size, lipid composition, and physical characteristics.2–4 Furthermore, liposomes can entrap both hydrophobic and hydrophilic drugs and are able to continuously release the entrapped substrate,5 thus being useful drug carriers.6–8 Polyethylene glycol (PEG) modification on the liposomal surface is known to be effective in preventing their uptake by the reticuloendothelial system (RES). Incorporation of PEG-lipids causes the liposome to remain in the blood circulation for extended periods of time (ie, t½ > 40 hours) and distribute through an organism relatively evenly with most of the dose remaining in the central compartment (ie, the blood) and only 10% to 15% of the dose being delivered to the liver.9–11 Long-circulating, PEGylated liposomes provide an attractive platform to improve the therapeutic index of a variety of drugs. A number of drugs have already been successfully encapsulated in liposomes, from antibacterials12 and interferons13 to antitumor drugs such as doxorubicin.14

Albendazole (ABZ), methyl [5-(propylthio)-1-H-benzimidazol-2yl] carbamate, is a benzimidazol derivative and it is effective in the treatment of echinococcosis, hydatid cysts, and neurocysticercosis. The therapeutic response of albendazole (20% to 50%) in cases of echinococcosis is variable and difficult to predict.15,16Albendazole’s poor intestinal absorption (<5%) (due to its low aqueous solubility) is probably a major determinant of the variable response rate.17 In vitro and in vivo antifungal activity of albendazole was studied by Hardin and colleagues.18 Albenza® (albendazole) is an orally administered broad-spectrum anthelmintic and has been used for hydatid disease with promising results.19–21 Repeated dose therapy is the only treatment for this disease and a course of albendazole for prescribed period has to be

completed by the patients. Therefore, preparation of drug formulations that facilitate controlled release of the drug to the target site is an important goal.

The objective of the present study was to prepare albendazole-encapsulated liposomal formulations. A comparison study was performed between conventional and PEG-coated or stealth liposomes to evaluate thein vitro performance of these formulations. Characterization of the prepared liposomes regarding physical morphology, particle size, and in vitro drug release was performed. A stability study was performed to investigate the release of drug from liposomes during storage.

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Materials and methodsMaterials

Albendazole was purchased from Cipla Pharma Co. (Mumbai, India). Egg phosphatidylcholine (PC), cholesterol (CH), and PEG were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the chemicals and solvents used in the study were of analytical and high-pressure liquid chromatography (HPLC) grade and purchased from Sigma Chemicals Co. (St. Louis, MO, USA), Himedia Laboratories Ltd. (Mumbai, India), Bangalore Genei (Bengalaru, India), and deionized water was used throughout the experiments.

Methods

Preparation of conventional and stealth liposomes

The PC liposomes were prepared using a rapid evaporation method.22 The composition of the conventional liposomes prepared was albendazole-encapsulated PC:CH taken in the ratio of 6:4 and dissolved in a chloroform:methanol mixture (1:1, V/V). The PEG-liposomes were prepared by mixing albendazole-encapsulated PC:CH:PEG-PC in the ratio of 5:4:1. Lipids were dissolved in 50 mL of isopropyl methanol:chloroform solution taken in the ratio of 1:1. Albendazole was dissolved in the same solvent mixture and added to the lipid solution. The mixture was sonicated for four minutes in a bath-type sonicator (Soniprep 150; MSE, Van Nuys, CA, USA). The organic solvent was then removed using rotary evaporator at 40°C and 50 rpm (Metrex, Delhi, India). In the case of albendazole, the resulting thin lipid film was slowly hydrated using bicarbonate buffer (pH 9.0). The process of hydration was carried out by rotation at a low speed at 55°C in a rotary evaporator under atmospheric pressure followed by hand shaking for 15 minutes at 55°C in a thermostatically controlled water bath. The resulting liposomal dispersion was left to mature overnight at 4°C. All the above steps were performed under aseptic conditions. All glassware was sterilized by autoclaving, and the entire procedure was performed in a laminar flow hood (Lab Companion; JEIO Tech, Seoul, Korea).

Free drug separation

Free unentrapped drug was separated from albendazole-encapsulated conventional and stealth liposomes by centrifugation at 20,000 g for 1 hour at 4°C in a refrigerated centrifuge (model 3–18 K; Sigma-Aldrich). The pellets formed were washed twice with 10 mL bicarbonate buffer (pH 9.0) and recentrifuged again for 1 hour.

Determination of encapsulation efficiency

The percentage of drug encapsulated was determined after lysis of the prepared liposomes with absolute alcohol and sonication for 10 minutes.23 The concentration of albendazole in absolute alcohol was determined spectrophotometrically at 265 nm using a UV-visible spectrophotometer (model UV-1700 (E); Schimadzu, Kyoto, Japan) in triplicate. The encapsulation efficiency expressed as entrapment percentage was calculated through the following relationship:24

Encapsulation efficiency % =Total drug−free drugTotal drug ×100.

Characterization of albendazole iposomes

Liposomes vary in size from 20 nm to several μm. Using a transmission electron microscope, size of conventional and PEG-coated liposomes were determined. The liposomes were photographed at an original magnification ×1000 to ×25000, using a transmission electron microscope (TEM; JEOL, Tokyo, Japan).

Stability study

The stability of the albendazole-encapsulated PEG-liposomes was evaluated after storage at −20°C, +4°C and 25°C for three months. The particle size distribution and drug encapsulation efficiency of the samples were determined as a function of the storage time.

In vitro drug release studies

Drug release from liposomes was studied using a dialysis method. Dialysis bags were soaked before use in distilled water at room temperature for 12 hours to remove the preservative, followed by rinsing thoroughly in distilled water. In vitro release of albendazole from liposomes was conducted by dialysis in a dialysis sac (12,000 MW cut off; Sigma-Aldrich) with 150 mL of phosphate-buffered saline (PBS; pH 5.6) containing 7% (V/V) propylene glycol and 25% (V/V) methanol at 37°C following the method published previously.25 Three sacs were prepared containing free albendazole as control, albendazole in conventional liposomes, and albendazole in PEGcoated liposomes.

Liposomal concentrate (equivalent to 2 mg albendazole) dispersed in 1 mL of bicarbonate buffer (pH 9) was placed in a dialysis bag. Control bags were prepared and tested along with the liposomal dispersions. Each control bag contained 2 mg albendazole. Two ends of the dialysis sac were tightly bound with threads. The sac was hung inside a conical flask with the help of a glass rod so that the portion of the dialysis sac with the formulation dipped into the buffer solution. The flask was kept on a magnetic stirrer (Matrex) and stirring was maintained at 100 rpm at 37°C with a thermostatic control. Samples were collected every half an hour over a period of five hours and assayed spectrophotometrically for drug content at 295.7 nm.

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Results and discussion

Albendazole-encapsulated liposomes

Drug-encapsulated PEG-liposomes were prepared by the combination of PC:CH:PEG-PC in the ratio of 5:4:1. On the other hand, conventional liposomes were prepared by mixing PC:CH in the ratio of 6:4 and lipids were dissolved in methanol:chloroform solution taken in the ratio (1:1

V/V). TEM micrograph was taken at 20,000 and 25,000 magnifications and clearly showed the formation of liposomes of moderate sizes ranging 50 to 150 nm. (Figures 1a and and1b).1b). Most of the liposomes formed appeared spherical or slightly asymmetrical in shape and were mainly unilamellar in arrangement.

Figure 1Transmission electron microscopic photograph of liposome A) Albendazole-loaded non-PEGylated or conventional liposomes and B) Albendazole-loaded PEGylated liposomes.

Encapsulation efficiency

The absorbance at different concentration of albendazole was measured using a UV-vis spectrophotometer at 295.7 nm. The standard curve of the albendazole was made by plotting absorbance against the concentration (Figure 2). The absorbance is no longer linear at concentration 10–200 μg/mL and 400–500 μg/mL. The concentration of the nonencapsulated drug in the supernatant was determined using this curve. Consequently, the encapsulation efficiency of different types of liposomes entrapping albendazole was easily calculated. The encapsulation efficacy was obtained as the mass ratio between the amount of the drug incorporated in liposomes and this ratio was used in the liposome preparation. By inspection of Table 1, it is obvious that albendazole-encapsulation efficiency had higher values in cases of PEGylated liposomes than in conventional liposomes. The total drug used during the preparation of liposomes was 1 mL of 1 mM albendazole. So using the formula the encapsulation efficiency of non-PEGylated and albendazole-loaded PEGylated liposomes was found to be 72% and 81%, respectively.

Figure 2The standard curve of Albendazole drug at 295.7 nm.

Table 1Encapsulation efficiency of liposomes

The highest encapsulation efficiency (81%) was observed with the PEGylated liposomes composed of PC:CH:PEG-PC (5:4:1) molar ratio. Liposomes prepared with PC:CH (7:2) molar ratio, showed the lowest encapsulation efficiency and were excluded from further studies.

Absorbance at various concentrations of albendazole was measured using a UV-vis spectrophotometer at 295.7 nm. The standard curve for each drug release was established using the Beer–Lambert equation, which correlates the extinction coefficient (ɛ) of a substance with the absorbance (A): A = ɛ.c.b where: ɛ is extinction coefficient; c is concentration of solution (mol/L); b is thickness (cm).

Stability study

PEGylated liposomes were selected and their physical and chemical stability was evaluated at three different temperatures for 3 months (Table 2). Initially, the mean vesicle size was 150 nm and encapsulation efficiency was 81%, no significant changes in drug encapsulation efficiency was observed during the course of stability study for formulations stored at −20°C and 4°C (P > 0.05) but there was a significant decrease in albendazole encapsulation efficiency for liposomes stored at room temperature (P < 0.05). The mean vesicle size showed an increase at all storage temperatures (P < 0.05) while particle size of the formulations was increased up to fourfold after three months at room temperature. This extraordinary increase in the particle size of liposomes may be due to the aggregation or swelling of liposomes. Further experiments are required to explain this phenomenon. Results of the stability study are shown in (Figure 3).

Figure 3Albendazole encapsulated PEGylated liposomes stability after 3 months storage at three different temperatures.

Table 2Particle size of albendazole-loaded conventional and PEGylated liposomes mean size (nm)

In vitro release studies

Evaluation of in vitro drug release from encapsulated liposome was done by dialysis method. The output obtained by the dialysis method provided a correlation with the in vivo release.26 The in vitro release behavior of the free albendazole, albendazole-loaded conventional liposomes, and PEGylated liposomes, is summarized in the cumulative percentage release shown in Figure 4. For the measurement of release rate of the free albendazole, and from the liposomes both PEG coated and uncoated, was measured at 37°C in PBS containing 7% (V/V) PEG and 25% (V/V) methanol. Over a period of five hours the measurement was taken after every half an hour at 295.7 nm. The value of concentration corresponding to the absorbance was calculated from the albendazole standard curve. The drug release rate of conventional liposomes and PEGylated liposomes or stealth liposomes is often comparable.

Figure 4In vitro release curve of albendazole -loaded non-PEGylated, PEGylated liposomes and free drug in phosphate buffer at 37°C.

The release rate of the drug was highest for the free drug while the rate of release from PEG liposomes was less than that of conventional liposomes. It seems that by adding PEG to the liposomes, the release rate of drug from the liposomes may decrease in its values. This confirms the fact that PEG acts as a barrier against diffusion of hydrophilic drugs. The hydrophobic long alkyl chains of the polymer may act as a barrier and the drug was effectively entrapped in the polymers.

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Discussion

Among a variety of targeted drug carrier systems, liposomes have been studied extensively because of their capability to accommodate a large variety of drugs, alongside their good biocompatibility, low toxicity, and lack of immune system activation or suppression.27

The present study was designed to develop and compare albendazole containing nanovesicular conventional and PEGylated liposomes. Comparing the results of both conventional and PEGylated liposomal formulations of the same composition and molar ratio, it is obvious that PEGylated liposomes showed a more sustained action owing to the presence of PEGcoating on the surface, which release the drug slowly over a prolonged period of time. It is to be noted that the in vitro release results are consistent with those of the encapsulation efficiency, as the

PEGylated liposomes with the highest cholesterol content PC:CH:PEG-PC (5:4:1) molar ratio and the highest encapsulation efficiency (ie, low leakage ability) showed the lowest drug release percentage. There was no significant difference between stability results of the liposome storage at 4°C or −20°C. The results of the stability study revealed that the prepared liposomes are stable for more than three months’ storage at 4°C or −20°C. This extraordinary increase in the particle size of liposomes at room temperature may be due to the aggregation or swelling of liposomes

As a rule of thumb, the drug concentration in the sink phase in release experiments should be kept below 10% saturation. If the drug is poorly soluble in water, nonaqueous solvents or solubilizing agents may be added to the sink. In case of albendazole-encapsulated liposomes, a release medium containing 7% (V/V) PEG and 25% (V/V) methanol was employed to provide sink conditions. The reproducibility and efficacy of the release study were ensured through a control sample containing drug in the free form. This could ensure that the dialysis membrane was not a barrier throughout the release study. The free albendazole (control sample) was released in about two hours.

The in vitro release behavior of free albendazole, albendazole-loaded conventional liposomes and PEGylated liposomes was summarized. In case of free albendazole more than 80% drug was released within the first sampling time (30 minutes) while both liposomal formulations produced an initial slower effect in which albendazole release was more than 25% for PEGylated and 35% for non-PEGylated liposomes within the first sampling time (30 minutes). The literature reports that drug release profiles from liposomes characteristically show an initial fast drug loss followed by slower rates of drug loss.28,29 The initial fast rate of release is commonly ascribed to drug detachment from liposomal surface while the later slow release results from sustained drug release from the inner lamellae. Albendazole is mainly associated within the bilayer lipid structure of the liposomes. The in vitro release study of albendazole showed no burst effect so that the drug transport out of the liposomes was driven mainly by a diffusion-controlled mechanism. The slight deviation at 210 minutes seems to be the effect of agglomeration of more PEG over liposome surface after a particular time interval which reshuffles soon it self due to intermolecular interaction with the surrounding environment.

Compared to the control data, albendazole release from PEGylated liposomes is prolonged. The slower release in PEGylated liposomes could be because of the fast hydration process at presenting the PEG on the surface of the particles. This result suggests that it takes time for albendazole to be released once encapsulated in the liposomes because lipid bilayer is stabilized by cholesterol. Thus a depot effect could be achieved using liposomes, especially in the PEGylated liposomal formulation. The above results, which suggest that the drug would be stable in the blood circulation and would be released slowly at the target site, are indications that our PEGylated liposomal formulation meets the requirements for an effective drug delivery system.30 Also the drug release data confirmed the drug entrapment efficiency results determined.

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Conclusion

Evaluation of in the vitro release profile of hydrophobic drugs from liposomal formulations could be problematic. But this could be manipulated through employment of a proper release

medium that could provide sufficient sink conditions without affecting the stability of the liposomal formulation. The results suggest that albendazole released for a prolonged period of time from the PEGylated liposomal formulations. Since they had nanodimensions, longer residence time in systemic circulation could help them reaching the target tissues. Nanosized PEGylated liposomes, would be a promising delivery systems for albendazole in the treatment of hydatid cyst. However, further studies including in vivo experiments are warranted.

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