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The present study was purported to formulate nonionic surfactant vesicles niosomes with cholesterol, using different molar ratio of span 40, span 60 and cholesterol, by ether injection method. The aim of this study was to design and determine the in vitro and stability study of niosome containing naproxen for its controlled release behavior. For this purpose formulation were optimized and characterized for particle size, particle shape, entrapment efficiency, in vitro drug release study and also kept for stability studies at 4°C, 25°C and 37°C in thermostatic oven for a month. Controlled release behavior observed by taking the consideration on % entrapment efficiency. The result for particle size of optimized niosomal formulations was found to be in the range of 5.34±0.45-9.7±0.84 µm. The vesicles were smooth, spherical and multilamellar in shape. The maximum % drug entrapment and % cumulative drug release was determined (88.44±0.28% and 87.88±0.66% respectively) in case of span 60 formulation after 10 h. The stability study suggest 4°C as optimum storage temperature. In conclusion the entrapment efficiency, in vitro release studies and stability studies shows controlled release behavior of drug from the niosome stable at 4°C when it prepared with span 60.
Surfactant, Cholesterol, Stability study.
In the past, for the development of novel drug delivery system (NDDS) significant interest has been made. The novel drug delivery system should ideally fulfill two basics. Firstly, it should channel the active entity to the site of action. Secondly, it should deliver the drug at a required rate by the wants of the body, over the period of treatment. Some dosage forms unable to meet one of these requirements such as Conventional dosage forms including prolonged release dosage forms. Currently, no available drug delivery system behaves perfectly, but sincere attempts have been made to achieve them through various novel approaches . A number of non-ionic surfactants have been used to prepare vesicles viz. polyglycerol alkyl ethers , glucosyl dialkyl ethers , crown ethers, esterlinked surfactants, poly oxyethylene alkyl ether, brij and series of spans and tweens [5,6 ] and the resultant vesicles have been termed niosomes. High patient compliance as compared to oily dosage forms is reported when vesicular suspension prepared in aqueous vehicle. They possess an infrastructure consisting of hydrophilic and lipophilic moieties together and as a result can accommodate drug molecules with a wide range of solubility. The vesicle formulation characteristics are variable as well as controllable. When altering the vesicle composition, size, lamellarity, surface charge, tapped volume, and concentration, the vesicle characteristics can be controlled. Vesicles release the drug in controlled manner and may acts as a depot . Other advantage of niosomes includes: these are osmotically active and stable, increase the stability of entrapped drug. The handling and storage of surfactants requires no special conditions. These improve oral bioavailability of poorly absorbed drugs and also enhance the skin penetration of drugs. These may be fabricated to reach the site of action by oral, parenteral as well as topical routes. The surfactants are biodegradable, biocompatible and non-immunogenic . Transdermel drug delivery offers many advantages over traditional methods. As a substitute for the oral route, avoidance of gastrointestinal absorption by transdermal route, with its associated pitfalls of enzymatic and pH associated deactivation. Also allows for reduced pharmacological dosing due to the shortened metabolizing pathway of the transdermal route as comparison to the gastrointestinal pathway . Some properties such as Release of the medicament from the vehicle, Penetration through the skin barrier and Activation of the pharmacological response which influence the transdermal drug delivery .
For successfully developing a transdermal drug delivery system,
the drug should be chosen with great care by taking a consideration of some desirable physiochemical properties of drug like molecular weight should have a less than approximately 1000 daltons, should have affinity for both - lipophilic and hydrophilic phases, low partitioning coefficient and drug should have low melting point. Along with these properties the drug should be potent, having short half life . That is the reason behind chosen the naproxen because it fulfills the maximum requirements which is essential for the transdermal drug delivery.
Naproxen is a nonsteroidal anti-inflammatory drug (NSAID) commonly used for the alleviation of pain, fever, inflammation and stiffness caused by conditions such as osteoarthritis, kidney stones, rheumatoid arthritis, psoriatic arthritis, gout, ankylosing spondylitis, menstrual cramps, tendinitis, bursitis . It is also used for the treatment of primary dysmenorrhea . It works by inhibiting both the COX-1 and COX-2 enzymes . Naproxen metabolized and excreted through the hepatic and renal, respectively. Naproxen is a member of the 2-arylpropionic acid (profen) family of NSAIDs. The free acid is an odorless, white to off-white crystalline substance. It is lipid-soluble and practically insoluble in water [11,12].
Material and methods
Naproxen (NPX) was a supplied as gift sample from Ranbaxy Research Labs, Gurgaon, India. Triton X-100, Span 40, Span 60 and Cholesterol were purchased from Sigma St Louis, MO, USA. Diethyl ether and methanol were purchased from E Merck, Mumbai, India. The materials were used as received and were of analytical grade.
Preparation of niosomes
Ether injection method provides a means of making niosomes with slide modification. The surfactant and cholesterol in different molar ratio, viz. 0.5:1, 1:1, 1.5:1, 2.0:1 and 1.0:2 (Table 1) were dissolved in 20 mL of diethyl ether, thereafter injected slowly through a 14 gauge needle into an aqueous solution (maintained at 60°C) of 1% Naproxen (NPX) . The vaporization of ether leads to formation of single layered vesicles [13,14].
Table I Composition of niosomal formulations
ratio (μ mol)
Size and shape of niosomes
The size of niosomes was determined using a light microscope (BEM-21, Besto Microscope, India) fitted with an ocular micrometer and stage micrometer. Scanning electron microscopy (SEM) (Philips-XL-20, Netherlands) was performed to characterize the surface morphology of the formed niosomes. Niosomes were mounted directly onto the sample stub and coated with gold film (200 nm) under reduced pressure (0.133 Pa) .
The niosomal dispersions were centrifuged (90 XL Ultracentrifuge, Beckman, USA) at 10,000 - g for 20 min to separate unentrapped drug and washed with phosphate buffered saline (pH 7.4). The clear supernatant was analyzed for naproxen by UV-spectrophotometer (Shimadzu UV-1700, Japan) at 226 nm. The amount of entrapped drug (eq. 1) was obtained by subtracting amount of unentrapped drug from the total drug incorporated .
In vitro release study
The niosomes encapsulating naproxen were separated by gel filtration on sephadex G-50 column which was kept in double distilled water for 10 h for swelling. Then the prepared niosomal suspension (1mL) was placed on the top of the column and elusion was carried out using normal saline. The niosomes loaded with naproxen elutes out first as a slightly dense, white opalescent suspension, were followed by free drug. Separated niosomes were filled in a dialysis tube to which a dialysis sac was attached to either end. The dialysis tube was suspended in phosphate buffered saline (pH 7.4), stirred with a magnetic stirrer and samples were withdrawn at specific time intervals and analyzed spectrophotometrically. The volume was replenished with the same amount of fresh dissolution fluid each time to maintain the sink condition .
The optimized Niosomal formulations were subjected to stability studies by storing at 4°C, 25°C and 37°C in thermostatic oven for a month . Then drug content of all the formulations was determined.
Results and discussion
Preparation of niosomes
Niosomes are non-ionic surfactant based vesicles and Self assembly of non-ionic surfactant in aqueous media results in closed bilayer structure. Except surfactant bilayer, niosomes carry all the properties of liposomes.
Size and shape of niosomes
Particle size determination of niosomes was performed by optical microscope after storage. The various ratios of all niosomal formulations were taken for size analysis. The vesicle size of the niosomes was found to be in the range of 5.34±0.45-9.7±0.84 µm. The size of the vesicles was uniform and independent of surfactant.
The surface morphology of niosomes was determined by scanning electron microscopy (SEM). The niosomes were smooth, spherical in shape and mostly small multilamellar (Figure 1). The vesicles were isolated and separate with no aggregation or agglomeration.
Figure 1 Scanning electron micrograph (SEM) of span 60 formulation
The maximum percent drug entrapment (88.44±0.28%) was observed with span 60 (Figure 2). Increase in cholesterol with surfactant concentration, resulted in increased percent drug entrapment and further increase in cholesterol concentration did not showed any influence on percent drug entrapment. From the above study it was observed that as the cholesterol content in the vesicles increased, the incorporation of the drug in the vesicles also increased . Cholesterol improves the fluidity of the bilayer membrane and also improves the stability of bilayer membrane in the presence of biological fluids such as blood/plasma (Table 2).
Table 2 % Entrapment efficiency (% EE) of different niosomal formulations of span 40 and span 60 containing naproxen
Figure 2 % Entrapment efficiency (% EE) of different niosomal formulations of span 40 and span 60 containing naproxen
In vitro release study
The in vitro release studies showed that time taken for drug release was 10 h for niosomes containing naproxen. The maximum % cumulative drug release observed with the niosomes prepared using span 60. As the cholesterol ratio increased the % cumulative drug release also increased but afterwards it shows diminished release of drug  (Table 3). The slow release of drug from multilamellar vesicles may be ascribed to the fact that multilamellar vesicles consist of several concentric sphere of bilayer above the aqueous compartment (Figure 3).
Table 3 In vitro release studies of different formulations of span 60 containing naproxen
% Cumulative drug release
Figure 3 In vitro release studies of different formulations of span 60 containing naproxen
The stability study exposed that the % entrapment efficiency of niosome formulation after storage for one month at 4°C, 25°C and 37°C. The % entrapment efficiency values upon storage were 96.65±0.45%, 90.53±0.71% and 85.32±0.49% at 4°C, 25°C and 37°C, respectively (Table 4). The % drug entrapment of formulations stored at 4°C was highest followed by the formulation stored at 25°C and 37°C. This may be due to phase transition of surfactants and lipid causing leakage from vesicle at higher temperature during storage. Hence, from the data, the optimum storage condition for the naproxen niosomes was found to be 4°C. Non-ionic surfactant with cholesterol is a suitable carrier for the preparation of niosomes of naproxen. Stability study reveals that span 60 showed maximum % drug entrapment efficiency which can be attributed to high lipophilicity of the surfactants. However, higher concentration of drug targeted to various sites may help in the reduction of dose required for therapy and thereby dose related systemic side effects too .
Table 4 Stability studies of different formulations of span 60 containing naproxen
The niosomal formulations containing naproxen using span 60 had particle size, particle shape, entrapment efficiency, in vitro drug release profile and stability at 4°C rendering these non-ionic surfactant vesicles suitable for topical application. The niosomes as compared to liposome represent a significant improvement in transdermal delivery of bioactives by eliminating physical stability problems, such as aggregation or fusion of vesicles and leaking of entrapped drugs during long-term storage and releases the drug in a rate controlled manner for a period of time. Thus niosomes are superior in terms of convenience of storage, transport and dosing as compared to conventional dosage forms. The study warrants extensive animal studies in order to establish the therapeutic benefits of vesicular carriers in transdermal application.