Niosomes are non-ionic surfactant-based bilayer membrane vesicles that are formed by self-assembly upon hydration. Non-ionic surfactants are amphiphilic molecules that are biodegradable, biocompatible, and non-immunogenic. Niosomes are capable of entrapping both hydrophobic and hydrophilic drugs [1
]. They have been successfully manufactured for oral delivery of cytotoxic agents such as paclitaxel [2
] at a lower cost with various surfactant combinations by film hydration method. The potential use of niosomes as an oral delivery system has been studied to overcome the challenges of limited absorption due to poor drug stability and poor water solubility in the gastrointestinal tract [3
]. Niosomes are similar to liposomes (which are comprised of phospholipids) as drug delivery systems. Unlike liposomes, niosomes have less chemical instability problems, but they are associated with physical stability issues such as fusion, aggregation, sedimentation, and drug leakage during storage [3
]. Numerous studies have shown that niosomes have a high potential as a carrier for poorly water-soluble drugs such as paclitaxel [2
], valsartan [5
], candesartan [6
], lornoxicam [7
], diacerein [8
], griseofulvin [9
], flurbiprofen [10
], and diclofenac [11
Spans are the product name marketed for sorbitan esters; they are produced by the dehydration of sorbitol. The hydrophile-lipophile balance (HLB) value of Span decreases with increasing the length of the alkyl chain (increasing the number of fatty acid groups); for example, sorbitan monostearate (Span® 60) has an HLB value of 4.7 and sorbitan tristearate (Span® 65) has an HLB value of 2.1; sorbitan monooleate (Span® 80) has an HLB value of 4.3, and sorbitan trioleate (Span® 85) has an HLB value of 1.8.
Their gel transition temperature increases as the length of the acyl chain increases resulting in decreased leakage of drugs from niosomes [12
]. All Spans have the same head group and different alkyl chain length [13
]. As the alkyl chain length increases, the entrapment efficiency of hydrophobic drugs is expected to increase. The encapsulation efficiency (%EE) is decreasing from Span®
), and Span®
80 (unsaturated C18
). This was in an agreement with flurbiprofen pro-niosomes studied by Mokhtar et al. [10
]. In addition, the study found that with increasing total lipid or drug concentration use resulted in an increased %EE of the flurbiprofen produced. Sorbitan monostearate (Span®
60) with a C18
chain has a gel transition temperature of 56–58 °C and an HLB value of 4.7, exhibits the highest entrapment efficiency, therefore it was chosen for this current study. The molar ratio of cholesterol incorporated with surfactants may affect the entrapment of drugs into niosomes. Nasseri [14
] reported that the equimolar mixture (Span®
and cholesterol) represented the critical composition as there is only one hydrogen bonding group on the cholesterol moiety to interact with oxygen functionalities on the Span®
60, resulting in an increase in membrane cohesion.
The incorporation of cholesterol with surfactants of lower HLB values has shown to promote the gel liquid transition temperature of the vesicle [15
]. Kumar and Rajeshwarrao [12
] reported the addition of cholesterol enables more hydrophobic surfactants (lower HLB values) to form niosomes by suppressing the tendency of aggregation. Cholesterol helps by increasing the orientation order of their relatively mobile hydrocarbon chains of liquid-crystalline phospholipid bilayers, decreasing bilayer permeability and reducing the efflux of the entrapped drug. This resulted in prolonged drug retention [16
] and effectively prevented leakage of the drug from niosomes [13
Other additive agents to enhance the characteristics of the niosome vesicles are co-surfactants. Co-surfactants are usually more hydrophilic; therefore, they act as emulsifying, solubilizing, and wetting agents as they have generally a higher HLB value from 12 to 16 and a higher molecular weight of over 1000 Da. Co-surfactants are commonly used in the literature for niosome formulations, for example Cremophor®
ELP (purified polyoxyl 35 castor oil) [17
RH40 (hydrogenated polyoxyl 40 castor oil) [18
], and Solutol®
HS15 (polyoxyl 15 hydroxystearate) [19
HS15 is known to inhibit P-glycoprotein, which is an ATP-dependent pump that is responsible for reducing drug intestinal absorption by efflux transportation. Addition of this agent has shown to enhance paclitaxel aqueous solubility and permeability across Caco-2 monolayer cell without inducing cytotoxicity [17
]. This would further enhance oral bioavailability of drugs such as cinnarizine (in this study) in the niosomal formulations.
There are numerous studies which have investigated the preparation of niosomes using the conventional thin-film hydration method and the reverse phase evaporation technique. The formation of film by evaporation of organic solvent is followed by hydration to produce multi-lamellar vesicles which are non-reproducible and a post size reduction process is needed to generate homogenous vesicles [18
]. Compared to the conventional bulk method, microfluidic technique has been used to prepare lipid-based nanoparticles and liposomes to generate reproducible small-sized nanoparticles for drug encapsulation. Correia et al. [19
] reported that microfluidic systems step up in the area of drug delivery with promising features that allow control of particle size and increased stability of the final liposome products. Hence, this technique will be used in this current research for niosome production.
Cinnarizine is a piperazine derivative with antihistaminic, sedative, antiserotonergic, antidopaminergic, and calcium channel blocking activities [20
]. It is also used as cerebral blood flow improver in the management of various peripheral and cerebral vascular disorders. Cinnarizine is a lipophilic weak base (literature pKa values of 1.95 and 7.47) and a log P
of 5.6 [21
]. The drug is practically insoluble in water in its unionized form and has a narrow absorption window. After oral administration of conventional tablet formulations, absorption is relatively slow, peak serum concentrations occurring after 2.5 to 4 hours [22
] from the upper gastrointestinal tract. This is due to patients’ gastric acidity influence on the dissolution and absorption of cinnarizine.
The study was aimed to develop and characterize cinnarizine-containing niosomes prepared by thin-film hydration and microfluidic methods, with the future aim to coat the niosome vesicles manufactured with a muco-adhesive agent (chitosan) for a prolonged drug retention in the stomach (to enhance drug bioavailability). To the best of our knowledge, there are no literature available for formulation of cinnarizine in niosome systems using either thin-film hydration or microfluidic methods, hence it is worth to characterize niosomes with this hydrophobic drug.
2. Experimental Section
Cinnarizine, Span® 60 (S60), cholesterol (Cho), phosphate buffered saline (PBS) tablets, chloroform, ethanol, hydrochloric acid 37%, and isopropanol were purchased from Sigma-Aldrich (Dorset, UK). Cremophor® ELP (ELP), Cremophor® RH40 (RH40), and Solutol® HS15 (HS15) were obtained from BASF (Cheshire, UK). All materials and chemicals were of analytical grade and used as received.
2.2. Preparation of Niosomes
In conventional thin-film hydration method, weighed lipids (refer to Table 1
) and cinnarizine were dissolved in chloroform and then transferred into a 100 mL round-bottomed flask. The formation of film was produced by using the rotary evaporator (Buchi rotavapor R-210, Flawil, Switzerland) to remove chloroform under pressure of 325 ± 10 mbar at 60 °C in a water bath and 100 revolutions per minute (rpm). Afterwards, the thin film obtained was allowed to dry completely and cooled at room temperature before underwent hydration process with 20 mL of phosphate buffered saline (PBS 10 mM, pH 7.4) solution using water bath shaker at 60 °C and 100 rpm for 30 min to produce niosome suspensions. Size reduction step (e.g., sonication) was excluded to investigate the influence of preparation methods on niosome characteristics.
As for microfluidic method, NanoAssemblrTM
Benchtop system (Precision NanoSystems Inc., Vancouver, VAN, Canada) with a microfluidic cartridge and a heating block controller was used. Weighed lipids (refer to Table 1
) and cinnarizine (without cinnarizine for empty pre-formed niosomes) were dissolved in ethanol to be used as organic phase and aqueous phase used was PBS. Process parameters applied were flow rate ratio of 4:1 (aqueous:organic), a total flow rate of 12 mL/min, and at 60 ± 1 °C (these conditions have been used based on preliminary studies).
A modified dialysis method from reference [23
] was used for all niosome suspensions prepared to be dialysed (using a dialysis membrane, MWCO: 3500 Da) against 1 L of 0.1 M hydrochloric acid solution (HCl, pH 1.2) under magnetic stirring for overnight to remove non-entrapped free drug and any trace of organic solvent.
2.3. Morphological Analysis of Niosomes Using Optical Microscope
Freshly prepared niosome suspensions were observed under light microscope with magnification lens of 40× using a MicroCam Olympus BH-2/LB with AxioCam MRc (Carl ZEISS, Jena, Germany). The formations of niosomes were confirmed by observation under optical light microscope and real-time images were taken. The niosomes formed were all in a distinctive round, circular shape.
2.4. Fourier-Transform Infra-Red (FTIR) Spectroscopy
Purified niosome pellets obtained from centrifugation (15,000 rpm for 20 min at 4 °C) were re-dispersed with 5 mL deionised water (TFH-based niosomes) and 5% mannitol (MF-based niosomes), and then kept in an ultra-low freezer (−80 °C) for 2 h prior to the freeze-drying process using Christ Advance Alpha 2–4 LSCplus freeze dryer, Osterode, Germany. The vacuum was set to 0.035 mbar with ice condenser set at −81 °C and shelf temperature at 10 °C. The infrared spectra of individual material and freeze-dried niosome samples were taken using a Shimadzu IRAffinity-1S spectrophotometer (Shimadzu UK Ltd., Buckinghamshire, UK). The spectra were recorded using 12 scans in the wavelength range (4000–550 cm−1) with a resolution of 4 cm−1 to study their possible interactions using the Shimadzu LabSolutions IR. Under the same conditions, the infrared spectrum of the cinnarizine pure drug was also taken for identification of its principle functional groups.
2.5. Thermal Characteristics of Niosomes
A standard mode DSC conditioning was performed at 75 °C for 120 min hold time without a refrigerated cooling system (RCS) (DSC Q1000 TA Instruments, Ghent, Belgium) with empty cell chamber. Afterwards, temperature calibration was carried out using pure indium with the RCS. All freeze-dried niosome samples (refer to Section 2.4
) weighed between 2 and 8 mg using Mettler MT5 balance (Mettler Toledo, Leicester, UK) were placed within standard aluminium hermetic pans and lids for DSC runs. DSC runs were performed at a heating rate of 10 °C/min from 25–300 °C. The nitrogen gas flow rate was 50 mL/min with the RCS on. Thermal Universal Analysis 2000 was used to perform analysis of the obtained DSC thermograms.
2.6. Particle Size, Polydispersity, and Charge of Niosomes
An aliquot from each of the freshly prepared niosome suspensions was used for measurement of particle size, polydispersity index (PDI), and zeta potential (ZP) in 1/20 dilution with deionised water, using Malvern Zetasizer Nano ZSP (Cambridge, UK). Measurement angle of 173° backscatter was used for angle of measurement detection. Three measurements were taken for each sample. Hydrodynamic size and PDI were measured using dynamic light scattering (DLS), and the zeta potential was determined using laser Doppler electrophoresis. The PDI is an indicator for particle size distribution ranging from 0 (narrow distribution) to 1 (wide distribution).
2.7.1. Calibration Curve
Cinnarizine standard solutions (10 to 500 ng/mL pure cinnarizine in 0.1 M HCl solution, pH 1.2) were prepared for calibration measurement using reverse phase ultra-high performance liquid chromatography (uHPLC). The HPLC system (Agilent technologies, Waldbronn, Germany) consisted of Agilent Chem Station LC-DAD with UV detector. The method was developed using Agilent Zorbax Eclipse Plus C18 (50 mm × 2.1 mm × 1.8 µm) column with gradient system of mobile phases consisting of 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B (see Table 2
). The flow rate was maintained at 0.3 mL/min. The column temperature was maintained at 40 °C and the detection wavelength used at 255 nm. The injection volume used was 10 µL for all measurements (standard solutions and samples). Agilent ChemStation software was used to record and integrate responses of peak areas and retention time.
2.7.2. Drug Encapsulation Efficiency and Drug Release Study
Purified niosome suspensions and their respective dialysates were assayed for the encapsulation efficiency, %EE. Isopropanol was used to disrupt the purified niosomes and filtered using 0.45 µm syringe filter prior to HPLC measurements for quantification. %EE was calculated as the percentage of total drug content (entrapped cinnarizine) after excluding free drug (non-entrapped cinnarizine) and based on the calibration equation obtained (Figure 1
A modified method [24
] for in vitro drug release study was performed using dialysis membrane containing 5 mL of the purified niosome suspension (MF-based S60:Cho:ELP) immersed in a beaker containing 200 mL of 0.1 M HCl solution (pH 1.2) as dissolution medium. The beaker was placed in a water bath shaker at 37 °C and 50 rpm. At predetermined intervals, 1 mL of the dissolution medium was withdrawn and then replaced with 1 mL of fresh 0.1 M HCl solution. The drug contents in the dissolution medium were calculated using the HPLC measurements based on the calibration equation obtained.