http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.873837
RESEARCH ARTICLE
Nonionic surfactant-based vesicular system for transdermal drug delivery Saeed Ghanbarzadeh1,2,3, Arash Khorrami1,3,4, and Sanam Arami1,3,5 Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 2Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 3Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 4Department of Pharmacology & Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, and 5Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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Abstract
Keywords
Objective: The objective of this study was to formulate and evaluate the Ibuprofen niosomal formulation as a transdermal drug delivery system. Materials and methods: Niosomes were prepared by a modified ethanol injection method, using Span 60, Tween 60 and Tween 65 as well as cholesterol with various cholesterol:surfactant molar ratios. The prepared vesicles were characterized for entrapment efficiency (EE), particle size, zeta potential and in vitro release study. Skin permeation studies were conducted using modified Franz diffusion cell, and excised rat skin was treated with niosomal, liposomal and conventional Carbopol 914 gel of Ibuprofen. Results and discussion: The results showed that the type of surfactant and molar ratio of cholesterol:surfactant altered the EE, size and in vitro drug release of niosomes. Higher EE was obtained with the niosomes prepared with cholesterol and Span 60 at molar ratio of 0.5:1. It has been observed that both niosomal and liposomal formulations enhanced the drug permeation and the percentage of accumulated dose in the skin compared to control conventional gel formulation. However, niosomes prepared by Span 60 and Tween 65 exhibited higher permeation and retention of Ibuprofen, respectively. Conclusion: Our results suggested that niosomal formulations could be used as a promising carrier for the Ibuprofen transdermal delivery system.
Ibuprofen, niosome, nonionic surfactant, skin permeation, transdermal drug delivery
Introduction Transdermal drug delivery represents an attractive alternative to oral and parenteral drug delivery routes, which has been proven beneficial in reducing dose frequency, achieving target delivery, easily administrating and avoiding hepatic first pass metabolism. The therapeutic effect of percutaneous formulations depends not only on the action of the drug itself but also on other structural factors of the vehicle (Knepp et al., 1990; Barry, 2001; Wokovich et al., 2006). Nano sized carriers have been receiving special interest with the aim of minimizing the side effects and improving effectiveness of drug therapy. Using colloidal particulate carrier-based drug delivery systems such as liposomes, ethosomes, transfersomes and niosomes has distinct advantages over conventional dosage forms, because these particles can act as drug containing reservoirs as well as penetration enhancer (Irwin et al., 1990; Fang et al., 2001a; Balakrishnan et al., 2009; Ghanbarzadeh &
Address for correspondence: Sanam Arami, Department of Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98 (411) 3372256. Fax: +98 (411) 3344798. Email: [email protected]
History Received 12 October 2013 Revised 16 November 2013 Accepted 29 November 2013
Arami, 2013a,b). Niosomes are nonionic surfactant vesicles that can encapsulate drug molecules with a wide range of solubility and possess potential to delivery of hydrophobic and hydrophilic drugs (Uchegbu & Vyas, 1998; Pham et al., 2012). Niosomes similar to liposomes are biodegradable, biocompatible and non-immunogenic and exhibit flexibility in their structural characterization. Niosomes have been investigated for drug delivery through the most common routes of administration, such as intramuscular, intravenous, subcutaneous, ocular, oral and transdermal. Due to their ability of carrying a great range of drugs, these lipid vesicles have been widely used in various drug delivery systems like targeting and controlled releasing (Sˇentjurc et al., 1999; Watson et al., 2012; Elhissi et al., 2013; Kong et al., 2013). Niosomes have higher chemical stability as a result of the incorporation of the surfactants, which are not easily hydrolyzed or oxidized like phospholipids which are due to the presence of esteric bonds. On the other hand, low reproducibility, due to the use of phospholipids, variable purity of phospholipids in liposomes and high cost have led scientist to search for vesicles prepared from other materials, such as nonionic surfactants. However, they are also some problems related to their physical stability including fusion, aggregation, sedimentation and leakage (Hu & Rhodes, 1999;
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Murdan et al., 1999; Rentel et al., 1999; Kumar & Rajeshwarrao, 2011). Niosomes increase the permeation of drugs across skin via two proposed mechanisms either by the penetration enhancer effect of the nonionic surfactants or vesicle–skin interactions. Due to the penetration of phospholipid molecules or nonionic surfactants into the lipid layers of the stratum corneum and epidermis, vesicular formulations may act as penetration enhancer and facilitate dermal delivery leading to higher localized drug concentrations. They also serve as a local depot for the sustained release of dermal active compounds including antibiotics, corticosteroids, minoxidil, finasteride or retinoic acid (Fang et al., 2001a; Manconi et al., 2006; Balakrishnan et al., 2009; Manosroi et al., 2010; Ammar et al., 2011; Kong et al., 2013; Muzzalupo et al., 2011). Nonionic surfactants are the most common type of surfactants used in the preparing of vesicles, due to the superiority over other counterparts in the case of stability, compatibility and toxicity aspects. Sorbitan fatty acid esters (SpanTM) are the most generally used surfactants found in literature (Ghanbarzadeh & Arami, 2013c). All Span types have the same head group and different alkyl chain length. A number of researchers also investigated TweensTM as nonionic surfactants in niosomes formations. Tweens are polysorbates derived from PEGylated sorbitan esterified with fatty acids (Fang et al., 2001b; Manosroi et al., 2003; Hua & Liu, 2007; Liu et al., 2007; Di Marzio et al., 2011; Kumar & Rajeshwarrao, 2011; Sakai et al., 2011; Junyaprasert et al., 2012). Ibuprofen is a non-steroidal anti-inflammatory drug. The mechanism of action of the Ibuprofen is primarily due to the inhibition of prostaglandin biosynthesis through the inhibition of the cyclooxygenase (COX) enzymes, COX-l and COX-2. It is widely approved for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, gout, extraarticular disorders, bursitis, tendonitis and non-articular rheumatic condition. Its oral administration is associated with severe adverse effects in the gastrointestinal tract such as epigastric pain, heartburn, nausea, diarrhea, vomiting, peptic ulcer and hepatic impairment (Saville, 2001; Yong et al., 2004; Newa et al., 2007). Although the skin is generally recognized for its effective barrier properties compared to
other biological membranes, transdermal drug delivery has been recognized as an alternative route for oral delivery of Ibuprofen. Different strategies have been used to increase the local bioavailability of topically administered Ibuprofen. In order to increase the therapeutic efficacy of topically administered drugs, it is necessary to employ percutaneous absorption enhancer and appropriate vehicles (Brown et al., 2001; Santi et al., 2003; Chen et al., 2006; Godwin et al., 2006; Rhee et al., 2008). Hence, in this study, attempts have been made to enhance the transdermal permeation of Ibuprofen using niosomal formulation incorporated in CarbopolÕ 914 gel form.
Materials and methods Materials Ibuprofen was received as a gift from Zahravi pharmaceutical Company (Tabriz, Iran). Span 60, Tween 60, Tween 65, soya lecithin and cholesterol were purchased from Merck Company (Darmstadt, Germany). All chemicals and solvents were analytical or HPLC grade. Fresh distilled water was used for preparing the formulations. Preparation of vesicles In this study, a modified ethanol injection method was used for the preparation of both Ibuprofen niosomes and liposomes (Ghanbarzadeh et al., 2013b). Lecithin was used for the preparation of liposomes, and three different surfactants including Span 60, Tween 60 and Tween 65 were used for the preparation of niosomes. Three different cholesterol:surfactant ratios (0:1, 0.5:1 and 1:1) were taken for formulation of niosomes (Table 1). The drug concentration was kept constant in all formulations. Briefly, precisely weighed amounts of cholesterol, drug and surfactants or lecithin, in the case of niosome or liposome, respectively, were dissolved in absolute ethanol for each batch. The obtained solutions were then taken in a syringe and injected dropwise into the phosphate buffered saline (PBS) pH 7.4 (ethanol:PBS, 1:10) at 50 C under mixing by homogenizer (8000 rpm) for 1 h. As the lipid solution was injected slowly into the aqueous phase,
Table 1. Compositions, entrapment efficiency percent (EE%), particle size (PS), polydispersity index (PI), zeta potential and in vitro released drug over 24h (D24h) of formulations. Formulation (code)
EE% SD
PS (nm) SD
PI
Zeta potential (mV) SD
D24h (%) SD
N1 (Chol:Span 60, 0:1) N2 (Chol:Span 60, 0.5:1) N3 (Chol:Span 60, 1:1) N4 (Chol:Tween 60, 0:1) N5 (Chol:Tween 60, 0.5:1) N6 (Chol:Tween 60, 1:1) N7 (Chol:Tween 65, 0:1) N8 (Chol:Tween 65, 0.5:1) N9 (Chol:Tween 65, 1:1) L1 (Chol:PC, 0:1) L2 (Chol:PC, 0.5:1) L3 (Chol:PC, 1:1) C1 Carbopol gel C2 Hydroethanolic solution
58.3 2.3 65.2 1.2 59.8 2.9 46.3 0.9 50.3 3.1 45.2 2.6 54.4 1.9 58.1 3.6 52.1 1.8 51.3 4.6 55.6 3.6 49.6 2.8 – –
165.3 10.3 182.3 20.2 135.6 12.5 298.6 30.4 286.3 26.8 315.6 15.9 231.3 24.3 215.6 19.8 264.3 33.2 265.3 17.6 211.6 16.3 298 42.1 – –
0.23 0.29 0.19 0.31 0.25 0.36 0.29 0.22 0.30 0.31 0.32 0.35 – –
17.2 1.3 15.3 2.6 16.3 2.4 12.5 3.2 13.6 1.6 19.2 2.1 10.2 0.9 16.2 1.1 17.2 2.0 5.3 0.09 6.3 1.0 8.2 0.8 – –
84.90 5.3a 76.22 6.2a 71.63 3.9a 60.05 4.8 56.27 8.2 51.42 3.7 75.66 4.6a 61.20 5.5 65.80 6.8a 57.97 8.3 56.82 3.2 48.29 4.6 55.3 2.3 62.3 1.8
Results were presented as mean standard deviation (n ¼ 3). p50.05 compared to C1.
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DOI: 10.3109/10717544.2013.873837
vaporization of ethanol resulted in the vesicles formation (Ghanbarzadeh et al., 2013d). Size distribution and zeta potential Mean vesicle size as well as polydispersity index (PI) of niosomes and liposomes were determined using Shimadzu particle size analyzer (WingSALD-2101, Tokyo, Japan). The average particle size was measured in triplicate. Zeta potential of Ibuprofen-loaded vesicles was determined using Zetasizer (Malvern Instruments, Malvern, UK). Analysis time was kept 60 s, and the average zeta potential of the vesicles was determined.
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Entrapment efficiency The prepared vesicular dispersions were subjected for dialysis method for 24 h in 10 C (lower than glass transmission temperature of nonionic surfactants) to separate unentrapped drug. Vesicles were disrupted by ethanol and analyzed by validated HPLC method (Al-Saidan, 2004) to calculate the amount of entrapped drug according to following equation (Ghanbarzadeh et al., 2013b): entrapped drug Entrapment efficiency ð%Þ ¼ 100 total drug added Stability studies Physical stability tests of the prepared vesicles were carried out to investigate the vesicle aggregation and drug leakage from vesicles during storage period. The prepared Ibuprofenloaded vesicles were stored in transparent vials covered with plastic cap at 4 C and ambient temperature for three months. The physical stability was evaluated by determining mean vesicle size, zeta potential and entrapment efficiency percent (EE%) over a three-month period. Samples from each batch were withdrawn at definite time intervals, and the particle size as well as residual amount of the drug in the vesicles were determined as described previously after separation from unentrapped drug (Ghanbarzadeh et al., 2013c). Preparation of Ibuprofen vesicular gels The Ibuprofen-loaded liposomes and niosomes (separated from the unentrapped drug) were incorporated into a gel base containing CarbopolÕ 914. Briefly, vesicular dispersion was dispersed in the gel containing 10% CarbopolÕ 914 with gentle stirring to obtain the Ibuprofen gel (5%, w/w). A fixed concentration of Ibuprofen was used in all formulations to make the vehicles effect on percutaneous absorption comparable. In vitro release of Ibuprofen from vesicles Ibuprofen release from niosomes and liposomes was determined using the membrane diffusion technique. One gram of niosomal or liposomal gel (equivalent to 50 mg of Ibuprofen) was placed in a dialyzer covered with polycarbonate membrane (cutoff: 100 nm). Subsequently, the dialyzer was suspended in the beaker containing PBS pH 7.4 (100 mL) and constantly stirred at 50 rpm at 37 1 C. Two milliliter samples were withdrawn at predetermined time intervals and
Niosomes as transdermal drug delivery system
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replaced with equal volume of fresh and prewarmed PBS (Ghanbarzadeh et al., 2013a). The Ibuprofen concentration in the samples was measured, and the obtained data were analyzed to determine the drug release profile over 24 h. In vitro skin permeation study Rat skin permeation of Ibuprofen from various gel formulations was studied using modified Franz diffusion cell having the area between the donor and the receiver chamber of 2.51 cm2 and the volume of the receptor compartment of 20 mL. The receptor compartment containing PBS (pH 7.4) was constantly stirred magnetically at 100 rpm and at controlled temperature of 37 2 C during the experiment. The skin was prepared from the abdominal skin of male Wistar rats (150–200 g and 8–10 weeks of age). The rat skins were shaved, excised and the subcutaneous fat was carefully removed and finally washed with saline solution. To kept same condition for all excised skins, skins were kept for saturation in PBS for 6 h before using for permeation studies. This helped the skins to be hydrated similarly. The skin was mounted with the subcutaneous side facing upwards to the donor compartment and subsequently, accurately weighed different formulations (amount equivalent to 100 mg of drug) were applied on the skin and spread by means of a spatula (ensuring that there were no air bubbles between the formulation and donor surface) and covered with paraffin film. Samples in the receiver chamber were obtained over 24 h and analyzed using HPLC method. The cumulative amount of the permeated Ibuprofen per unit of skin was plotted versus time. The flux was calculated from the slope of linear part of the amount of the drug permeated versus time curve. The permeability coefficient (Kp) indicates the penetrating power of a substance through the stratum corneum and was calculated by the following equation: Kp ¼ J=C where J is the flux and C is the initial concentration of formulation in donor compartment. To determine the remaining drug, the formulation was removed from the skin surface, skin was cut into small pieces, soaked and shaken in 1.2 mL of benzethonium hydroxide for 24 h for lysis the skins. For sedimentation of skin proteins, 100 mL trichloroacetic acid 10% was added and centrifuged in 6000 rpm. Finally, the Ibuprofen in the skin was extracted by absolute ethanol under sonication, centrifuged at 1500 rpm and subsequently the drug content was assayed. The amount of Ibuprofen was measured and expressed as percentage of initially applied drug. All determinations were carried out in triplicate (Ghanbarzadeh & Arami, 2013a). This study was conducted in accordance to the Guide lines of the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health Publication No 85-23, revised 1985). Statistical analysis The data have been reported as mean SD (n ¼ 3), and statistical analysis of the data was carried out by SPSS software (SPSS Inc., Chicago, IL) using one way analysis of variance test at a level of significance of p50.05.
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Results and discussions
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Characteristics of the vesicles Amongst many reported methods for the preparation of niosomes or liposomes, ethanol injection method was selected as it is the most used method with greater EE% and smaller particle size without need for extrusion (Pons et al., 1993; Maitani et al., 2007). Vesicles size, PI and zeta potential of vesicles were presented in Table 1. Polydispersity index of the samples was ranged from 0.2 to 0.35. The size distribution of the niosomal formulations showed a narrow peak, indicating that this method generated a relatively homogeneous vesicles. The particle size range was found to be 135–315 nm for all formulations. The size of the vesicles is dependent on the HLB value of the surfactant used and hydrophilic monomers increase the vesicle surface energy and water uptake into the bilayer, which causes vesicle enlargement (Manosroi et al., 2003; Balakrishnan et al., 2009; Kumar & Rajeshwarrao, 2011; Mahale et al., 2012). Therefore, the lower HLB, the smaller size of the vesicles. The cholesterol:surfactant ratio had no significant effect on particle size of vesicles. Entrapment efficiency Data shown in Table 1 revealed that the entrapment efficiencies for prepared niosomes using span 60 were superior to those prepared using Tween 60 and Tween 65. This may be due to the fact that hydrophilic surfactants owing to high aqueous solubility do not form proper vesicular structure in aqueous medium, whereas due to lipophilic nature of surfactant with lower HLB, they form vesicles and entrap the lipophilic or amphiphilic drugs. Therefore, Ibuprofen encapsulation efficiency (EE%) was decreased while increasing the surfactant HLB value. Literature review also revealed that the lower the HLB, the higher the amount of lipophilic drug, which can be incorporated into the niosomal bilayers
(Yoshioka et al., 1994; Vora et al., 1998; Manconi et al., 2006; Pardakhty et al., 2007; Mahale et al., 2012). Table 1 also revealed that in both liposomal and niosomal formulations, while increasing cholesterol, the hydrophobicity and stability of the bilayer were increased similar to EE%. In vitro drug release The in vitro release profile of all formulations was studied using dialysis tube and represented in Figure 1. The in vitro release study revealed that the cumulative percent release was maximum for the formulations containing Span 60 (Table 1). Table 1 also indicated that the increasing of cholesterol:surfactant proportion to more than 0.5, decreased leakage of bilayer structure and released drug over 24 h. Increasing the cholesterol proportion led to more rigid lipid bilayers as a barrier for drug release and decreased its leakage by improving the fluidity of the bilayer membrane and reducing its permeability, which led to lower drug release from the vesicles (Fang et al., 2001a; Devaraj et al., 2002; Manosroi et al., 2003; Nasseri, 2005). There was a significant difference in drug release rate between niosomal gel, liposomal gel and conventional Carbopol gel (p50.05). Physical stability study of Ibuprofen vesicles Physical stability study of the prepared vesicular dispersions was carried out for 90 d to investigate the particle size growth and drug leaching from niosomes during storage at refrigerator and room temperatures. The results showed higher percentage of drug retained in formulations stored at refrigerated conditions than room temperature after three months storage. This may be due to the higher fluidity of the bilayers at higher temperature resulting in higher drug leakage. Niosomes also were found to be reasonably stable in terms of aggregation, fusion and leakage compared to
Figure 1. The in vitro release profiles of all prepared formulations.
Niosomes as transdermal drug delivery system
DOI: 10.3109/10717544.2013.873837
liposomes. There was no significant difference in physical stability results between different niosomes stored at the same temperature conditions. Analysis of data obtained from drug leakage study revealed that niosomal drug gel was significantly more stable than niosomal suspension and also both formulations were significantly more stable at refrigerated temperature than room temperature (Table 2). The improved stability of niosomes after incorporation into gel base may be due to the prevention of niosomes fusion. Results suggested that keeping the niosomal product in refrigeration conditions minimized the instability problems. Skin permeation and disposition studies
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The cumulative amounts of permeated drug per unit area of rat skin, the fluxes and Ibuprofen permeability coefficient
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after 24 h from different niosomal and liposomal gel formulations were investigated using Franz diffusion cell and were presented in Figure 2 and Table 3. Table 3 also showed the percent of drug deposited after 24 h in rat skin from prepared gel formulations. Significant increase in the skin permeation of Ibuprofen has been observed with niosomal formulations compared to liposomal formulations and Carbopol gel. It was in this order: Span 604Tween 654Tween 604Liposome4Carbopol gel4Hydroethanolic solution. The maximum amount of Ibuprofen permeated over 24 h per unit area was found to be 14588.02 625.2 mg/cm2 in Span 60 niosomal gel. Accordingly, flux value obtained for Span 60 niosomal gel was maximum (608.6416 32.5 mg/cm2/h). The maximum amount of Ibuprofen permeated through skin and flux value of niosomal formulations was 2–4 folds higher than liposomal
Table 2. Physical stability of used formulation in skin permeation test during storage at 4 C and 25 C for three months. Particle size (nm) Formulation (code) N2 (Chol:Span 60, 0.5:1) 4 C 25 C N5 (Chol:Tween 60, 0.5:1) 4 C 25 C N8 (Chol:Tween 65, 0.5:1) 4 C 25 C L2 (Chol:PC, 0.5:1) 4 C 25 C
Zeta potential (mV)
EE%
Initial
After 3 months
Initial
After 3 months
Initial
After 3 months
182.3 20.2
196.6 12.3 225.3 18.6a
15.3 2.6
14.3 1.2 12.4 0.3
65.2 1.2
61.3 2.3 58.3 3.1
286.3 26.8
301.3 20.0 352.6 31.3a
13.6 1.6
13.1 0.9 12.2 1.1
50.3 3.1
50.1 1.9 42.3 1.2a
215.6 19.8
236.6 15.3 286.0 29.6a
16.2 1.1
15.2 1.3 12.9 0.8a
58.1 3.6
54.2 1.8 48.3 1.5a
211.6 16.3
231.0 16.3 253.6 28.3a
6.3 1.0
7.1 0.9 4.3 1.1a
55.6 3.6
53.2 0.8 48.6 1.0
Results were presented as mean standard deviation (n ¼ 3). p50.05 compared to L2.
a
Figure 2. The in vitro rat skin permeation profiles of Ibuprofen from different formulations. **p50.01 and ***p50.001 compared to L2 and ##p50.01 and ###p50.001 compared to C1.
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Table 3. Permeated amount of Ibuprofen at 24h, flux, permeability coefficient and residual drug remaining in the skin as percent of initially applied drug of the formulations.
Formulation (code) N2 (Chol:Span 60, 0.5:1) N5 (Chol:Tween 60, 0.5:1) N8 (Chol:Tween 65, 0.5:1) L2 (Chol:PC, 0.5:1) C1 Carbopol gel C2 Hydroethanolic solution
Permeated amount at 24 h (mg/cm2) a,b
14588.02 625.2 6797.196 536.3b,c 9117.514 542.3a,b 3563.63 452.2f 1926.39 51.2 867.23 36.2
Flux (mg/cm2/h) a,b
608.64 32.5 259.16 12.3b,c 380.40 23.2a,b 148.75 5.24f 82.06 3.69 37.77 2.68
Permeability coefficient (Kp) 103 (cm/h)
Residual drug (%)
a,b
12.17 2.13 5.18 0.89b,c 7.609 0.68a,b 2.971 1.52f 1.64 0.16 0.75 0.06
14.98 1.68b,c 9.10 1.24d,e 19.36 1.02a,b 6.62 2.38b 2.22 0.88 0.79 0.26
Results were presented as mean standard deviation (n ¼ 3). p50.05, cp50.01 and ap50.001 compared to L2 and ep50.05, fp50.01 and bp50.001 compared to C1.
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formulations. As can be observed in Table 2, vesicular formulations enhanced the Ibuprofen accumulation in rat skin. The maximum percentage of drug deposited in the rat skin was 19.366 1.02%, which belonged to Tween 65 niosomal gel, which was three times higher than liposomal gel. Therefore, results indicated that the formulated Ibuprofen niosomes noticeably enhanced skin permeation and were able to deposit the drug molecules in the skin, which was related to vesicle composition. Al-Saidan produced Ibuprofen saturated solutions using 0.1, 0.2, 0.3 and 0.4 M disodium hydrogen phosphate solution and the amount of permeated Ibuprofen increase up to 75000 mg/cm2 during 24 h. Irwin et al. also showed that addition of 1% of the dimethylamide as penetration enhancers for the transport of Ibuprofen from suspensions in 50% aqueous propylene glycol vehicles across rat skin can increase Flux from 25.2 up to 619.9 mmol/cm2/h. In the study by Bialik et al., incorporation of Brij 92 (2%) increased the permitted Ibuprofen up to 400 mg/cm2 from Ibuprofen gel (5%) (Bialik et al., 1990; Irwin et al., 1990; Iervolino et al., 2001; Al-Saidan, 2004). In another study, Iervolino et al. by increasing the concentration of PG could increase flux value more than two folds. Comparing our study results with previous investigations indicated that niosomal formulations are promising carriers for transdermal delivery of Ibuprofen.
Conclusion Ibuprofen was successfully entrapped within the niosomal vesicles with high efficiency. Vesicle size, EE and drug release were dependent on cholesterol:surfactant ratio as well as the kind of the surfactant used. In vitro permeation results of this study showed that composition of niosomes was very important for improving delivery and deposition of a lipophilic drug such as Ibuprofen. Nonionic surfactants not only are basis of niosomes but also have effective role in enhanced drug penetration through the skin. Flux value obtained for Span 60 niosomal gel was maximum, and on the other hand, Tween 65 (HLB: 10.5) niosomes have been shown to be able to greatly enhance drug retention, especially compared to the liposomal gel and Carbopol gel formulations of Ibuprofen. From a technical view, it is important to study the influence of various niosomal formulation variables, which need to be optimized for desired responses. The effects of wide range of surfactant also need to be studied. Overall, it can be concluded that
niosomes are promising carriers for transdermal delivery of Ibuprofen.
Acknowledgements The authors would like to thank the Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
Declaration of interest The authors report no declarations of interest.
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DOI: 10.3109/10717544.2013.873837
Ghanbarzadeh S, Arami S, Khorrami A. (2013a). Preparation of optimized Naproxen nano liposomes using response surface methodology. J Pharmaceut Invest [Epub ahead of print]. Ghanbarzadeh S, Valizadeh H, Zakeri-Milani P. (2013b). Application of factorial designs and response surface methodology in formulation development of sirolimus liposome prepared by thin film hydration technique. BioImpacts 3:75–81. Ghanbarzadeh S, Valizadeh H, Zakeri-Milani P. (2013c). The effects of lyophilization on the physico-chemical stability of sirolimus liposomes. Adv Pharm Bull 3:25–9. Ghanbarzadeh S, Valizadeh H, Zakeri-Milani P. (2013d). Sirolimus nano liposomes/optimization of sirolimus nano liposomes prepared by modified ethanol injection method using response surface methodology. Pharmind [Epub ahead of print]. Godwin DA, Wiley CJ, Felton LA. (2006). Using cyclodextrin complexation to enhance secondary photoprotection of topically applied ibuprofen. Eur J Pharm Biopharm 62:85–93. Hu C, Rhodes DG. (1999). Proniosomes: a novel drug carrier preparation. Int J Pharm 185:23–35. Hua W, Liu T. (2007). Preparation and properties of highly stable innocuous niosome in Span 80/PEG 400/H2O system. Colloids Surf Physicochem Eng Aspects 302:377–82. Iervolino M, Cappello B, Raghavan SL, Hadgraft J. (2001). Penetration enhancement of ibuprofen from supersaturated solutions through human skin. Int J Pharm 212:131–41. Irwin WJ, Sanderson FD, Li Wan Po A. (1990). Percutaneous absorption of ibuprofen and naproxen: Effect of amide enhancers on transport through rat skin. Int J Pharm 66:243–52. Junyaprasert VB, Singhsa P, Suksiriworapong J, Chantasart D. (2012). Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid. Int J Pharm 423:303–11. Knepp VM, Szoka Jr FC, Guy RH. (1990). Controlled drug release from a novel liposomal delivery system. II. Transdermal delivery characteristics. J Control Release 12:25–30. Kong M, Park H, Feng C, et al. (2013). Construction of hyaluronic acid noisome as functional transdermal nanocarrier for tumor therapy. Carbohydr Polym 94:634–41. Kumar GP, Rajeshwarrao P. (2011). Nonionic surfactant vesicular systems for effective drug delivery – an overview. Acta Pharmaceutica Sinica B 1:208–19. Liu T, Guo R, Hua W, Qiu J. (2007). Structure behaviors of hemoglobin in PEG 6000/Tween 80/Span 80/H2O niosome system. Colloids Surf Physicochem Eng Aspects 293:255–61. Mahale NB, Thakkar PD, Mali RG, et al. (2012). Niosomes: novel sustained release nonionic stable vesicular systems – an overview. Adv Colloid Interface Sci 183–4:46–54. Maitani Y, Igarashi S, Sato M, Hattori Y. (2007). Cationic liposome (DC-Chol/DOPE 1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression. Int J Pharm 342:33–9. Manconi M, Sinico C, Valenti D, et al. (2006). Niosomes as carriers for tretinoin: III. A study into the in vitro cutaneous delivery of vesicleincorporated tretinoin. Int J Pharm 311:11–9. Manosroi A, Khanrin P, Lohcharoenkal W, et al. (2010). Transdermal absorption enhancement through rat skin of gallidermin loaded in niosomes. Int J Pharm 392:304–10.
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Manosroi A, Wongtrakul P, Manosroi J, et al. (2003). Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids Surf B Biointerfaces 30:129–38. Murdan S, Gregoriadis G, Florence AT. (1999). Sorbitan monostearate/ polysorbate 20 organogels containing niosomes: a delivery vehicle for antigens? Eur J Pharm Sci 8:177–85. Muzzalupo R, Tavano L, Cassano R, et al. (2011). A new approach for the evaluation of niosomes as effective transdermal drug delivery systems. Eur J Pharm Biopharm 79:28–35. Nasseri B. (2005). Effect of cholesterol and temperature on the elastic properties of niosomal membranes. Int J Pharm 300:95–101. Newa M, Bhandari KH, Li DX, et al. (2007). Preparation, characterization and in vivo evaluation of ibuprofen binary solid dispersions with poloxamer 188. Int J Pharm 343:228–37. Pardakhty A, Varshosaz J, Rouholamini A. (2007). In vitro study of polyoxyethylene alkyl ether niosomes for delivery of insulin. Int J Pharm 328:130–41. Pham TT, Jaafar-Maalej C, Charcosset C, Fessi H. (2012). Liposome and niosome preparation using a membrane contactor for scale-up. Colloids Surf B Biointerfaces 94:15–21. Pons M, Foradada M, Estelrich J. (1993). Liposomes obtained by the ethanol injection method. Int J Pharm 95:51–6. Rentel CO, Bouwstra JA, Naisbett B, Junginger HE. (1999). Niosomes as a novel peroral vaccine delivery system. Int J Pharm 186:161–7. Rhee Y-S, Chang S-Y, Park C-W, et al. (2008). Optimization of ibuprofen gel formulations using experimental design technique for enhanced transdermal penetration. Int J Pharm 364:14–20. Sakai T, Kurosawa H, Okada T, Mishima S. (2011). Vesicle formation in mixture of a PEO-PPO-PEO block copolymer (Pluronic P123) and a nonionic surfactant (Span 65) in water. Colloids Surf Physicochem Eng Aspects 389:82–9. Santi P, Nicoli S, Colombo G, et al. (2003). Post-iontophoresis transport of ibuprofen lysine across rabbit ear skin. Int J Pharm 266:69–75. Saville DJ. (2001). Influence of storage on in-vitro release of ibuprofen from sugar coated tablets. Int J Pharm 224:39–49. Sˇentjurc M, Vrhovnik K, Kristl J. (1999). Liposomes as a topical delivery system: the role of size on transport studied by the EPR imaging method. J Control Release 59:87–97. Uchegbu IF, Vyas SP. (1998). Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int J Pharm 172:33–70. Vora B, Khopade AJ, Jain NK. (1998). Proniosome based transdermal delivery of levonorgestrel for effective contraception. J Control Release 54:149–65. Watson DS, Endsley AN, Huang L. (2012). Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens. Vaccine 30:2256–72. Wokovich AM, Prodduturi S, Doub WH, et al. (2006). Transdermal drug delivery system (TDDS) adhesion as a critical safety, efficacy and quality attribute. Eur J Pharm Biopharm 64:1–8. Yong CS, Oh Y-K, Jung SH, et al. (2004). Preparation of ibuprofenloaded liquid suppository using eutectic mixture system with menthol. Eur J Pharm Sci 23:347–53. Yoshioka T, Sternberg B, Florence AT. (1994). Preparation and properties of vesicles (niosomes) of sorbitan monoesters (Span 20, 40, 60 and 80) and a sorbitan triester (Span 85). Int J Pharm 105:1–6.
RESEARCH ARTICLE
Nonionic surfactant-based vesicular system for transdermal drug delivery Saeed Ghanbarzadeh1,2,3, Arash Khorrami1,3,4, and Sanam Arami1,3,5 Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 2Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 3Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, 4Department of Pharmacology & Toxicology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran, and 5Pharmaceutical Biotechnology Department, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
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Abstract
Keywords
Objective: The objective of this study was to formulate and evaluate the Ibuprofen niosomal formulation as a transdermal drug delivery system. Materials and methods: Niosomes were prepared by a modified ethanol injection method, using Span 60, Tween 60 and Tween 65 as well as cholesterol with various cholesterol:surfactant molar ratios. The prepared vesicles were characterized for entrapment efficiency (EE), particle size, zeta potential and in vitro release study. Skin permeation studies were conducted using modified Franz diffusion cell, and excised rat skin was treated with niosomal, liposomal and conventional Carbopol 914 gel of Ibuprofen. Results and discussion: The results showed that the type of surfactant and molar ratio of cholesterol:surfactant altered the EE, size and in vitro drug release of niosomes. Higher EE was obtained with the niosomes prepared with cholesterol and Span 60 at molar ratio of 0.5:1. It has been observed that both niosomal and liposomal formulations enhanced the drug permeation and the percentage of accumulated dose in the skin compared to control conventional gel formulation. However, niosomes prepared by Span 60 and Tween 65 exhibited higher permeation and retention of Ibuprofen, respectively. Conclusion: Our results suggested that niosomal formulations could be used as a promising carrier for the Ibuprofen transdermal delivery system.
Ibuprofen, niosome, nonionic surfactant, skin permeation, transdermal drug delivery
Introduction Transdermal drug delivery represents an attractive alternative to oral and parenteral drug delivery routes, which has been proven beneficial in reducing dose frequency, achieving target delivery, easily administrating and avoiding hepatic first pass metabolism. The therapeutic effect of percutaneous formulations depends not only on the action of the drug itself but also on other structural factors of the vehicle (Knepp et al., 1990; Barry, 2001; Wokovich et al., 2006). Nano sized carriers have been receiving special interest with the aim of minimizing the side effects and improving effectiveness of drug therapy. Using colloidal particulate carrier-based drug delivery systems such as liposomes, ethosomes, transfersomes and niosomes has distinct advantages over conventional dosage forms, because these particles can act as drug containing reservoirs as well as penetration enhancer (Irwin et al., 1990; Fang et al., 2001a; Balakrishnan et al., 2009; Ghanbarzadeh &
Address for correspondence: Sanam Arami, Department of Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98 (411) 3372256. Fax: +98 (411) 3344798. Email: [email protected]
History Received 12 October 2013 Revised 16 November 2013 Accepted 29 November 2013
Arami, 2013a,b). Niosomes are nonionic surfactant vesicles that can encapsulate drug molecules with a wide range of solubility and possess potential to delivery of hydrophobic and hydrophilic drugs (Uchegbu & Vyas, 1998; Pham et al., 2012). Niosomes similar to liposomes are biodegradable, biocompatible and non-immunogenic and exhibit flexibility in their structural characterization. Niosomes have been investigated for drug delivery through the most common routes of administration, such as intramuscular, intravenous, subcutaneous, ocular, oral and transdermal. Due to their ability of carrying a great range of drugs, these lipid vesicles have been widely used in various drug delivery systems like targeting and controlled releasing (Sˇentjurc et al., 1999; Watson et al., 2012; Elhissi et al., 2013; Kong et al., 2013). Niosomes have higher chemical stability as a result of the incorporation of the surfactants, which are not easily hydrolyzed or oxidized like phospholipids which are due to the presence of esteric bonds. On the other hand, low reproducibility, due to the use of phospholipids, variable purity of phospholipids in liposomes and high cost have led scientist to search for vesicles prepared from other materials, such as nonionic surfactants. However, they are also some problems related to their physical stability including fusion, aggregation, sedimentation and leakage (Hu & Rhodes, 1999;
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Murdan et al., 1999; Rentel et al., 1999; Kumar & Rajeshwarrao, 2011). Niosomes increase the permeation of drugs across skin via two proposed mechanisms either by the penetration enhancer effect of the nonionic surfactants or vesicle–skin interactions. Due to the penetration of phospholipid molecules or nonionic surfactants into the lipid layers of the stratum corneum and epidermis, vesicular formulations may act as penetration enhancer and facilitate dermal delivery leading to higher localized drug concentrations. They also serve as a local depot for the sustained release of dermal active compounds including antibiotics, corticosteroids, minoxidil, finasteride or retinoic acid (Fang et al., 2001a; Manconi et al., 2006; Balakrishnan et al., 2009; Manosroi et al., 2010; Ammar et al., 2011; Kong et al., 2013; Muzzalupo et al., 2011). Nonionic surfactants are the most common type of surfactants used in the preparing of vesicles, due to the superiority over other counterparts in the case of stability, compatibility and toxicity aspects. Sorbitan fatty acid esters (SpanTM) are the most generally used surfactants found in literature (Ghanbarzadeh & Arami, 2013c). All Span types have the same head group and different alkyl chain length. A number of researchers also investigated TweensTM as nonionic surfactants in niosomes formations. Tweens are polysorbates derived from PEGylated sorbitan esterified with fatty acids (Fang et al., 2001b; Manosroi et al., 2003; Hua & Liu, 2007; Liu et al., 2007; Di Marzio et al., 2011; Kumar & Rajeshwarrao, 2011; Sakai et al., 2011; Junyaprasert et al., 2012). Ibuprofen is a non-steroidal anti-inflammatory drug. The mechanism of action of the Ibuprofen is primarily due to the inhibition of prostaglandin biosynthesis through the inhibition of the cyclooxygenase (COX) enzymes, COX-l and COX-2. It is widely approved for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, gout, extraarticular disorders, bursitis, tendonitis and non-articular rheumatic condition. Its oral administration is associated with severe adverse effects in the gastrointestinal tract such as epigastric pain, heartburn, nausea, diarrhea, vomiting, peptic ulcer and hepatic impairment (Saville, 2001; Yong et al., 2004; Newa et al., 2007). Although the skin is generally recognized for its effective barrier properties compared to
other biological membranes, transdermal drug delivery has been recognized as an alternative route for oral delivery of Ibuprofen. Different strategies have been used to increase the local bioavailability of topically administered Ibuprofen. In order to increase the therapeutic efficacy of topically administered drugs, it is necessary to employ percutaneous absorption enhancer and appropriate vehicles (Brown et al., 2001; Santi et al., 2003; Chen et al., 2006; Godwin et al., 2006; Rhee et al., 2008). Hence, in this study, attempts have been made to enhance the transdermal permeation of Ibuprofen using niosomal formulation incorporated in CarbopolÕ 914 gel form.
Materials and methods Materials Ibuprofen was received as a gift from Zahravi pharmaceutical Company (Tabriz, Iran). Span 60, Tween 60, Tween 65, soya lecithin and cholesterol were purchased from Merck Company (Darmstadt, Germany). All chemicals and solvents were analytical or HPLC grade. Fresh distilled water was used for preparing the formulations. Preparation of vesicles In this study, a modified ethanol injection method was used for the preparation of both Ibuprofen niosomes and liposomes (Ghanbarzadeh et al., 2013b). Lecithin was used for the preparation of liposomes, and three different surfactants including Span 60, Tween 60 and Tween 65 were used for the preparation of niosomes. Three different cholesterol:surfactant ratios (0:1, 0.5:1 and 1:1) were taken for formulation of niosomes (Table 1). The drug concentration was kept constant in all formulations. Briefly, precisely weighed amounts of cholesterol, drug and surfactants or lecithin, in the case of niosome or liposome, respectively, were dissolved in absolute ethanol for each batch. The obtained solutions were then taken in a syringe and injected dropwise into the phosphate buffered saline (PBS) pH 7.4 (ethanol:PBS, 1:10) at 50 C under mixing by homogenizer (8000 rpm) for 1 h. As the lipid solution was injected slowly into the aqueous phase,
Table 1. Compositions, entrapment efficiency percent (EE%), particle size (PS), polydispersity index (PI), zeta potential and in vitro released drug over 24h (D24h) of formulations. Formulation (code)
EE% SD
PS (nm) SD
PI
Zeta potential (mV) SD
D24h (%) SD
N1 (Chol:Span 60, 0:1) N2 (Chol:Span 60, 0.5:1) N3 (Chol:Span 60, 1:1) N4 (Chol:Tween 60, 0:1) N5 (Chol:Tween 60, 0.5:1) N6 (Chol:Tween 60, 1:1) N7 (Chol:Tween 65, 0:1) N8 (Chol:Tween 65, 0.5:1) N9 (Chol:Tween 65, 1:1) L1 (Chol:PC, 0:1) L2 (Chol:PC, 0.5:1) L3 (Chol:PC, 1:1) C1 Carbopol gel C2 Hydroethanolic solution
58.3 2.3 65.2 1.2 59.8 2.9 46.3 0.9 50.3 3.1 45.2 2.6 54.4 1.9 58.1 3.6 52.1 1.8 51.3 4.6 55.6 3.6 49.6 2.8 – –
165.3 10.3 182.3 20.2 135.6 12.5 298.6 30.4 286.3 26.8 315.6 15.9 231.3 24.3 215.6 19.8 264.3 33.2 265.3 17.6 211.6 16.3 298 42.1 – –
0.23 0.29 0.19 0.31 0.25 0.36 0.29 0.22 0.30 0.31 0.32 0.35 – –
17.2 1.3 15.3 2.6 16.3 2.4 12.5 3.2 13.6 1.6 19.2 2.1 10.2 0.9 16.2 1.1 17.2 2.0 5.3 0.09 6.3 1.0 8.2 0.8 – –
84.90 5.3a 76.22 6.2a 71.63 3.9a 60.05 4.8 56.27 8.2 51.42 3.7 75.66 4.6a 61.20 5.5 65.80 6.8a 57.97 8.3 56.82 3.2 48.29 4.6 55.3 2.3 62.3 1.8
Results were presented as mean standard deviation (n ¼ 3). p50.05 compared to C1.
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DOI: 10.3109/10717544.2013.873837
vaporization of ethanol resulted in the vesicles formation (Ghanbarzadeh et al., 2013d). Size distribution and zeta potential Mean vesicle size as well as polydispersity index (PI) of niosomes and liposomes were determined using Shimadzu particle size analyzer (WingSALD-2101, Tokyo, Japan). The average particle size was measured in triplicate. Zeta potential of Ibuprofen-loaded vesicles was determined using Zetasizer (Malvern Instruments, Malvern, UK). Analysis time was kept 60 s, and the average zeta potential of the vesicles was determined.
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Entrapment efficiency The prepared vesicular dispersions were subjected for dialysis method for 24 h in 10 C (lower than glass transmission temperature of nonionic surfactants) to separate unentrapped drug. Vesicles were disrupted by ethanol and analyzed by validated HPLC method (Al-Saidan, 2004) to calculate the amount of entrapped drug according to following equation (Ghanbarzadeh et al., 2013b): entrapped drug Entrapment efficiency ð%Þ ¼ 100 total drug added Stability studies Physical stability tests of the prepared vesicles were carried out to investigate the vesicle aggregation and drug leakage from vesicles during storage period. The prepared Ibuprofenloaded vesicles were stored in transparent vials covered with plastic cap at 4 C and ambient temperature for three months. The physical stability was evaluated by determining mean vesicle size, zeta potential and entrapment efficiency percent (EE%) over a three-month period. Samples from each batch were withdrawn at definite time intervals, and the particle size as well as residual amount of the drug in the vesicles were determined as described previously after separation from unentrapped drug (Ghanbarzadeh et al., 2013c). Preparation of Ibuprofen vesicular gels The Ibuprofen-loaded liposomes and niosomes (separated from the unentrapped drug) were incorporated into a gel base containing CarbopolÕ 914. Briefly, vesicular dispersion was dispersed in the gel containing 10% CarbopolÕ 914 with gentle stirring to obtain the Ibuprofen gel (5%, w/w). A fixed concentration of Ibuprofen was used in all formulations to make the vehicles effect on percutaneous absorption comparable. In vitro release of Ibuprofen from vesicles Ibuprofen release from niosomes and liposomes was determined using the membrane diffusion technique. One gram of niosomal or liposomal gel (equivalent to 50 mg of Ibuprofen) was placed in a dialyzer covered with polycarbonate membrane (cutoff: 100 nm). Subsequently, the dialyzer was suspended in the beaker containing PBS pH 7.4 (100 mL) and constantly stirred at 50 rpm at 37 1 C. Two milliliter samples were withdrawn at predetermined time intervals and
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replaced with equal volume of fresh and prewarmed PBS (Ghanbarzadeh et al., 2013a). The Ibuprofen concentration in the samples was measured, and the obtained data were analyzed to determine the drug release profile over 24 h. In vitro skin permeation study Rat skin permeation of Ibuprofen from various gel formulations was studied using modified Franz diffusion cell having the area between the donor and the receiver chamber of 2.51 cm2 and the volume of the receptor compartment of 20 mL. The receptor compartment containing PBS (pH 7.4) was constantly stirred magnetically at 100 rpm and at controlled temperature of 37 2 C during the experiment. The skin was prepared from the abdominal skin of male Wistar rats (150–200 g and 8–10 weeks of age). The rat skins were shaved, excised and the subcutaneous fat was carefully removed and finally washed with saline solution. To kept same condition for all excised skins, skins were kept for saturation in PBS for 6 h before using for permeation studies. This helped the skins to be hydrated similarly. The skin was mounted with the subcutaneous side facing upwards to the donor compartment and subsequently, accurately weighed different formulations (amount equivalent to 100 mg of drug) were applied on the skin and spread by means of a spatula (ensuring that there were no air bubbles between the formulation and donor surface) and covered with paraffin film. Samples in the receiver chamber were obtained over 24 h and analyzed using HPLC method. The cumulative amount of the permeated Ibuprofen per unit of skin was plotted versus time. The flux was calculated from the slope of linear part of the amount of the drug permeated versus time curve. The permeability coefficient (Kp) indicates the penetrating power of a substance through the stratum corneum and was calculated by the following equation: Kp ¼ J=C where J is the flux and C is the initial concentration of formulation in donor compartment. To determine the remaining drug, the formulation was removed from the skin surface, skin was cut into small pieces, soaked and shaken in 1.2 mL of benzethonium hydroxide for 24 h for lysis the skins. For sedimentation of skin proteins, 100 mL trichloroacetic acid 10% was added and centrifuged in 6000 rpm. Finally, the Ibuprofen in the skin was extracted by absolute ethanol under sonication, centrifuged at 1500 rpm and subsequently the drug content was assayed. The amount of Ibuprofen was measured and expressed as percentage of initially applied drug. All determinations were carried out in triplicate (Ghanbarzadeh & Arami, 2013a). This study was conducted in accordance to the Guide lines of the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz-Iran (National Institutes of Health Publication No 85-23, revised 1985). Statistical analysis The data have been reported as mean SD (n ¼ 3), and statistical analysis of the data was carried out by SPSS software (SPSS Inc., Chicago, IL) using one way analysis of variance test at a level of significance of p50.05.
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Results and discussions
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Characteristics of the vesicles Amongst many reported methods for the preparation of niosomes or liposomes, ethanol injection method was selected as it is the most used method with greater EE% and smaller particle size without need for extrusion (Pons et al., 1993; Maitani et al., 2007). Vesicles size, PI and zeta potential of vesicles were presented in Table 1. Polydispersity index of the samples was ranged from 0.2 to 0.35. The size distribution of the niosomal formulations showed a narrow peak, indicating that this method generated a relatively homogeneous vesicles. The particle size range was found to be 135–315 nm for all formulations. The size of the vesicles is dependent on the HLB value of the surfactant used and hydrophilic monomers increase the vesicle surface energy and water uptake into the bilayer, which causes vesicle enlargement (Manosroi et al., 2003; Balakrishnan et al., 2009; Kumar & Rajeshwarrao, 2011; Mahale et al., 2012). Therefore, the lower HLB, the smaller size of the vesicles. The cholesterol:surfactant ratio had no significant effect on particle size of vesicles. Entrapment efficiency Data shown in Table 1 revealed that the entrapment efficiencies for prepared niosomes using span 60 were superior to those prepared using Tween 60 and Tween 65. This may be due to the fact that hydrophilic surfactants owing to high aqueous solubility do not form proper vesicular structure in aqueous medium, whereas due to lipophilic nature of surfactant with lower HLB, they form vesicles and entrap the lipophilic or amphiphilic drugs. Therefore, Ibuprofen encapsulation efficiency (EE%) was decreased while increasing the surfactant HLB value. Literature review also revealed that the lower the HLB, the higher the amount of lipophilic drug, which can be incorporated into the niosomal bilayers
(Yoshioka et al., 1994; Vora et al., 1998; Manconi et al., 2006; Pardakhty et al., 2007; Mahale et al., 2012). Table 1 also revealed that in both liposomal and niosomal formulations, while increasing cholesterol, the hydrophobicity and stability of the bilayer were increased similar to EE%. In vitro drug release The in vitro release profile of all formulations was studied using dialysis tube and represented in Figure 1. The in vitro release study revealed that the cumulative percent release was maximum for the formulations containing Span 60 (Table 1). Table 1 also indicated that the increasing of cholesterol:surfactant proportion to more than 0.5, decreased leakage of bilayer structure and released drug over 24 h. Increasing the cholesterol proportion led to more rigid lipid bilayers as a barrier for drug release and decreased its leakage by improving the fluidity of the bilayer membrane and reducing its permeability, which led to lower drug release from the vesicles (Fang et al., 2001a; Devaraj et al., 2002; Manosroi et al., 2003; Nasseri, 2005). There was a significant difference in drug release rate between niosomal gel, liposomal gel and conventional Carbopol gel (p50.05). Physical stability study of Ibuprofen vesicles Physical stability study of the prepared vesicular dispersions was carried out for 90 d to investigate the particle size growth and drug leaching from niosomes during storage at refrigerator and room temperatures. The results showed higher percentage of drug retained in formulations stored at refrigerated conditions than room temperature after three months storage. This may be due to the higher fluidity of the bilayers at higher temperature resulting in higher drug leakage. Niosomes also were found to be reasonably stable in terms of aggregation, fusion and leakage compared to
Figure 1. The in vitro release profiles of all prepared formulations.
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liposomes. There was no significant difference in physical stability results between different niosomes stored at the same temperature conditions. Analysis of data obtained from drug leakage study revealed that niosomal drug gel was significantly more stable than niosomal suspension and also both formulations were significantly more stable at refrigerated temperature than room temperature (Table 2). The improved stability of niosomes after incorporation into gel base may be due to the prevention of niosomes fusion. Results suggested that keeping the niosomal product in refrigeration conditions minimized the instability problems. Skin permeation and disposition studies
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The cumulative amounts of permeated drug per unit area of rat skin, the fluxes and Ibuprofen permeability coefficient
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after 24 h from different niosomal and liposomal gel formulations were investigated using Franz diffusion cell and were presented in Figure 2 and Table 3. Table 3 also showed the percent of drug deposited after 24 h in rat skin from prepared gel formulations. Significant increase in the skin permeation of Ibuprofen has been observed with niosomal formulations compared to liposomal formulations and Carbopol gel. It was in this order: Span 604Tween 654Tween 604Liposome4Carbopol gel4Hydroethanolic solution. The maximum amount of Ibuprofen permeated over 24 h per unit area was found to be 14588.02 625.2 mg/cm2 in Span 60 niosomal gel. Accordingly, flux value obtained for Span 60 niosomal gel was maximum (608.6416 32.5 mg/cm2/h). The maximum amount of Ibuprofen permeated through skin and flux value of niosomal formulations was 2–4 folds higher than liposomal
Table 2. Physical stability of used formulation in skin permeation test during storage at 4 C and 25 C for three months. Particle size (nm) Formulation (code) N2 (Chol:Span 60, 0.5:1) 4 C 25 C N5 (Chol:Tween 60, 0.5:1) 4 C 25 C N8 (Chol:Tween 65, 0.5:1) 4 C 25 C L2 (Chol:PC, 0.5:1) 4 C 25 C
Zeta potential (mV)
EE%
Initial
After 3 months
Initial
After 3 months
Initial
After 3 months
182.3 20.2
196.6 12.3 225.3 18.6a
15.3 2.6
14.3 1.2 12.4 0.3
65.2 1.2
61.3 2.3 58.3 3.1
286.3 26.8
301.3 20.0 352.6 31.3a
13.6 1.6
13.1 0.9 12.2 1.1
50.3 3.1
50.1 1.9 42.3 1.2a
215.6 19.8
236.6 15.3 286.0 29.6a
16.2 1.1
15.2 1.3 12.9 0.8a
58.1 3.6
54.2 1.8 48.3 1.5a
211.6 16.3
231.0 16.3 253.6 28.3a
6.3 1.0
7.1 0.9 4.3 1.1a
55.6 3.6
53.2 0.8 48.6 1.0
Results were presented as mean standard deviation (n ¼ 3). p50.05 compared to L2.
a
Figure 2. The in vitro rat skin permeation profiles of Ibuprofen from different formulations. **p50.01 and ***p50.001 compared to L2 and ##p50.01 and ###p50.001 compared to C1.
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Table 3. Permeated amount of Ibuprofen at 24h, flux, permeability coefficient and residual drug remaining in the skin as percent of initially applied drug of the formulations.
Formulation (code) N2 (Chol:Span 60, 0.5:1) N5 (Chol:Tween 60, 0.5:1) N8 (Chol:Tween 65, 0.5:1) L2 (Chol:PC, 0.5:1) C1 Carbopol gel C2 Hydroethanolic solution
Permeated amount at 24 h (mg/cm2) a,b
14588.02 625.2 6797.196 536.3b,c 9117.514 542.3a,b 3563.63 452.2f 1926.39 51.2 867.23 36.2
Flux (mg/cm2/h) a,b
608.64 32.5 259.16 12.3b,c 380.40 23.2a,b 148.75 5.24f 82.06 3.69 37.77 2.68
Permeability coefficient (Kp) 103 (cm/h)
Residual drug (%)
a,b
12.17 2.13 5.18 0.89b,c 7.609 0.68a,b 2.971 1.52f 1.64 0.16 0.75 0.06
14.98 1.68b,c 9.10 1.24d,e 19.36 1.02a,b 6.62 2.38b 2.22 0.88 0.79 0.26
Results were presented as mean standard deviation (n ¼ 3). p50.05, cp50.01 and ap50.001 compared to L2 and ep50.05, fp50.01 and bp50.001 compared to C1.
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d
formulations. As can be observed in Table 2, vesicular formulations enhanced the Ibuprofen accumulation in rat skin. The maximum percentage of drug deposited in the rat skin was 19.366 1.02%, which belonged to Tween 65 niosomal gel, which was three times higher than liposomal gel. Therefore, results indicated that the formulated Ibuprofen niosomes noticeably enhanced skin permeation and were able to deposit the drug molecules in the skin, which was related to vesicle composition. Al-Saidan produced Ibuprofen saturated solutions using 0.1, 0.2, 0.3 and 0.4 M disodium hydrogen phosphate solution and the amount of permeated Ibuprofen increase up to 75000 mg/cm2 during 24 h. Irwin et al. also showed that addition of 1% of the dimethylamide as penetration enhancers for the transport of Ibuprofen from suspensions in 50% aqueous propylene glycol vehicles across rat skin can increase Flux from 25.2 up to 619.9 mmol/cm2/h. In the study by Bialik et al., incorporation of Brij 92 (2%) increased the permitted Ibuprofen up to 400 mg/cm2 from Ibuprofen gel (5%) (Bialik et al., 1990; Irwin et al., 1990; Iervolino et al., 2001; Al-Saidan, 2004). In another study, Iervolino et al. by increasing the concentration of PG could increase flux value more than two folds. Comparing our study results with previous investigations indicated that niosomal formulations are promising carriers for transdermal delivery of Ibuprofen.
Conclusion Ibuprofen was successfully entrapped within the niosomal vesicles with high efficiency. Vesicle size, EE and drug release were dependent on cholesterol:surfactant ratio as well as the kind of the surfactant used. In vitro permeation results of this study showed that composition of niosomes was very important for improving delivery and deposition of a lipophilic drug such as Ibuprofen. Nonionic surfactants not only are basis of niosomes but also have effective role in enhanced drug penetration through the skin. Flux value obtained for Span 60 niosomal gel was maximum, and on the other hand, Tween 65 (HLB: 10.5) niosomes have been shown to be able to greatly enhance drug retention, especially compared to the liposomal gel and Carbopol gel formulations of Ibuprofen. From a technical view, it is important to study the influence of various niosomal formulation variables, which need to be optimized for desired responses. The effects of wide range of surfactant also need to be studied. Overall, it can be concluded that
niosomes are promising carriers for transdermal delivery of Ibuprofen.
Acknowledgements The authors would like to thank the Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran.
Declaration of interest The authors report no declarations of interest.
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