A comprehensive production method of self

International Journal of Pharmaceutics 486 (2015) 153–158

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

A comprehensive production method of self-cryoprotected nano-liposome powders Rikhav P. Gala a,b , Iftikhar Khan a , Abdelbary M.A. Elhissi c, ** , Mohamed A. Alhnan a, * a

School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom Vaccine Nanotechnology Lab Department of Pharmaceutical Sciences, School of Pharmacy, Mercer University, Atlanta, GA, USA c Pharmaceutical Sciences Section, College of Pharmacy, Qatar University, P.O. Box 2713, Doha, Qatar b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 21 November 2014 Received in revised form 16 March 2015 Accepted 17 March 2015 Available online 18 March 2015

This study provided a convenient approach for large scale production of hydrogenated soya phosphatidylcholine nano-liposome powders using beclometasone dipropionate as a model drug and sucrose as proliposome carrier. Fluid-bed coating was employed to manufacture proliposomes by coating sucrose with the phospholipid (5%, 10%, 15% and 20% weight gains), followed by hydration, size reduction using high pressure homogenization, and freeze-drying to yield stable nano-vesicles. High pressure homogenization was compared with probe-sonication in terms of liposome size, zeta potential and drug entrapment. Furthermore, the effect of freeze-drying on vesicle properties generated using both size reduction methods was evaluated. Results have shown that high-pressure homogenization followed by freeze-drying and rehydration tended to yield liposomes smaller than the corresponding vesicles downsized via probe-sonication, and all size measurements were in the range of 72.64–152.50 nm, indicating that freeze-drying was appropriate, regardless of the size reduction technique. The liposomes, regardless of size reduction technique and freeze drying had slightly negative zeta potential values or were almost neutral in surface charge. The entrapment efficiency of BDP in homogenized liposomes was found to increase following freeze-drying, hence the drug entrapment efficiency values in rehydrated liposomes were 64.9%, 57%, 69.5% and 64.5% for 5%, 10%, 15% and 20% weight gains respectively. In this study, we have reported a reliable production method of nano-liposomes based on widely applicable industrial technologies such as fluid-bed coating, high pressure homogenization and freeze-drying. Moreover, sucrose played a dual role as a carrier in the proliposome formulations and as a cryoprotectant during freeze-drying. ã 2015 Published by Elsevier B.V.

Keywords: Homogenizer Scale-up Asthma Cortisones Aerosol Inhalation

1. Introduction Liposomes are microscopic phospholipid vesicles which were used as artificial membrane models to mimic simple cell systems for the investigation of transport functions and mechanisms, and study the permeation properties of small ions and molecules, and the adhesion and fusion kinetics of biological cells. Since they were reported by Bangham and co-workers liposomes have attracted a lot of interest as drug carriers (Wagner and Vorauer-Uhl, 2011). Liposomes have been recognized as highly promising delivery carriers and many liposome formulations have gained clinical approval (Chang and Yeh, 2012) and many have reached the

* Corresponding author. Tel.: +44 (0)1772 893590. ** Corresponding author. Tel.: +974 (0) 4403 5632. E-mail addresses: [email protected], [email protected] (A.M.A. Elhissi), [email protected] (M.A. Alhnan). http://dx.doi.org/10.1016/j.ijpharm.2015.03.038 0378-5173/ ã 2015 Published by Elsevier B.V.

cosmetics market (Rahimpour and Hamishehkar, 2012), (Carbone et al., 2013) and (Choi and Maibach, 2005). Doxil1 and DaunoXome1 were the first liposome-based products to be commercially available for clinical use (Allen and Cullis, 2013). Many methods of liposome preparation on small laboratory scale level have been reported in the literature such as the reverse phase evaporation method (Patil and Jadhav, 2014). Sonication has been used widely for the production of nano-liposomes and nanoniosomes; however this approach is not applicable for large scale production and the use of titanium probe-sonicators may result in contamination of the liquid because of the titanium particles leaching from the probe into the preparation. Large scale production of liposomes has been proposed using solvent injection based methods such as improved supercritical reverse phase evaporation (ISCRPE) and depressurization of expanded solution into aqueous media (DESAM) (Meure et al., 2008). The inconsistency of the final product characteristics, low product yield, high cost and complexity of the methods are all obstacles hindering the

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development of new liposome products (Laouini et al., 2013). Micro-fluid methods for large-scale production of nano-liposomes have been proposed; however, this approach requires the design of special equipment and frequent replacement of the microengineered membranes (Laouini et al., 2013). Proliposomes have been proposed as a stable alternative with ability to generate liposomes upon addition of aqueous phase prior to administration. They have been conventionally made by coating carbohydrate carrier particles with phospholipids within a rotary evaporator (Elhissi and Taylor, 2005). Rare examples are available on the production of proliposomes via fluidized bed in larger quantities. Previous attempts used insoluble multilayer core (Chen and Alli, 1987) or ionisable substrate (Katare et al., 1990), which may compromised the stability of the hydrated formulation and limited its use. More recently, Tantisripreecha et al. (2012) used a coating pan to apply lipids as a sub-coat for enteric coating of protein tablets. Moreover, hydration of proliposomes predominantly generates micro-liposomes and liposome aggregates (Elhissi and Taylor, 2005), which may limit their potential for use clinically. Thus, the design of a comprehensive approach to manufacture stable nano-liposomes in larger quantity is highly in need. The aim of this study was to develop stable powdered phospholipid formulations that upon hydration can generate self-cryoprotected nano-liposomes. We have designed a production approach for liposomes based on manufacture of proliposomes via fluidized bed as an intermediate step. This was followed by hydration, high pressure homogenization and cryoprotected lyophilization, using the antiasthma steroid beclometasone dipropionate (BDP) as model drug. 2. Materials and methods 2.1. Materials Sucrose (AnalaR1 grade) was obtained from BDH Laboratory Supplies, UK. Hydrogenated soya phosphatidylcholine (HSPC) (Phospholipon1 90H) was a gift from Lipoid, Switzerland. Water, absolute ethanol, and methanol used for coating were all of HPLC grade and purchased from Fisher Scientific Ltd., UK. Beclometasone dipropionate (BDP) was of analytical grade and purchased from Sigma–Aldrich, USA. 2.2. Preparation of proliposomes Sucrose carrier particles were sieved using a sieving set (Fisher Scientific, UK) and particles in the size range of 400–800 mm were collected for the preparation of proliposomes. HSPC (10.6 g) was dissolved in absolute ethanol (200 mL) mixed using a magnetic stirrer at 60 C for 1 h to ensure complete lipid dissolution. BDP was incorporated to constitute 5 mole% of the lipid phase. Sucrose particles (40 g) were placed in the coating chamber of Strea1Classic fluidized bed-dryer (GEA Pharma systems, Switzerland) and coating with the ethanolic HSPC solution was carried out at the rate of 2.1 mL/min and atomization pressure of 0.3 bar. The inlet air temperature was maintained at 33 3 C and the outlet temperature was approximately 35 3 C. It was necessary to maintain the room temperature below 20 C to keep the temperature of the product relatively low during the coating process and avoid particle coalescence (i.e., agglomeration), since melting of lipids at high temperature may promote the adherence between the proliposome particles. In addition, low temperature may enhance the spreading of wet phospholipid on the sucrose carrier particles and is also highly desirable to provide better safety conditions during coating. Coating was completed within 2 h to obtain 5%, 10%, 15% and 20% weight gain from the original weight of sucrose used

prior to the commencement of coating. Fig. 1 summarizes the manufacturing steps carried out for the production of proliposomes and liposomes. 2.3. Preparation of liposomes from proliposomes Proliposome samples (150 mg) were hydrated using 3 mL of deionized water (65 C) in glass vials followed by vortex mixing for 2 min. The vials were placed in a water bath (Büchi B480, Switzerland) at 60 C for 15 min to ensure complete lipid hydration followed by further vortex mixing for 3 min. The liposomes were allowed to anneal for 2 h prior to further processing or analysis. 2.4. Scanning electron microscopy The proliposomes were fastened on a carbon pads (Agar Scientific, UK) and coated with gold using a JFC-1200 Fine Coater (JEOL, Tokyo, Japan). A scanning electron microscope (Quanta 200, FEI Company, USA) was utilized to observe the samples at 30 kV to provide details of the surface characteristics as well as the morphology of the uncoated and lipid-coated sucrose particles. 2.5. Size reduction of liposomes using probe-sonication or highpressure homogenization The hydrated liposome formulations were converted into nanoliposomes via size reduction using the Nano DeBEE high pressure homogenizer (BEE International, Inc., MA, USA). Liposomes generated from proliposome were placed into the sample holder and homogenization took place at a pressure of 25,000 psi at 20 C. All formulations underwent three cycles of homogenization in order to reduce particle size. The size of liposomes was measured using dynamic light scattering to ascertain the generation of nanoliposomes following the completed cycles of homogenization and in between the cycles. Size reduction of hydrated proliposomes was conducted using probe sonication (Sonics Vibra cell CV33, CT, USA) and the findings were compared to size analysed using high pressure homogenization. To minimize the risk of heat-induced sample degradation, the flask accommodating the sample was placed in an ice bath during probe-sonication at 50 W for 5 min. 2.6. Freeze-drying of liposome formulations The homogenized liposome formulations were loaded into vials and kept in the freezer ( 20 C) overnight. The vials were then loaded into the freeze-drier (Edwards, Micro Modulyo, IL, USA). The unit was closed and the temperature of the system was lowered to 20 C and vacuum of 1.5–2.0 mbar was applied for 8 h to obtain the dried liposomes. 2.7. Liposome size and zeta potential analysis The laser diffraction instrument Mastersizer Hydro 2000 SM (Malvern Instruments Ltd., UK) was used to measure the size distribution of liposomes prior to size reduction. The median size and size distribution were calculated by the instrument’s software as the volume median diameter (VMD; 50% undersize) and Span respectively, where Span = (90% undersize – 10% undersize)/VMD. Following size reduction, the size analysis of nano-liposomes was carried out via dynamic light scattering using Zetasizer Nano ZS (Malvern Instruments Ltd., UK). 2.8. Entrapment efficiency of BDP in liposomes The entrapment efficiency of BDP in sonicated or homogenized liposome vesicles was determined by adapting the method


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Fig. 1. Summary of method of production of liposome using the proliposomes technology.

previously used by Kumar et al. (2001). Briefly, proliposomes (100 mg) were hydrated with 5 mL of HPLC water and passed through syringe filter (0.45 mm). The BDP which was incorporated in the liposomes passed through the filter was considered as the entrapped percentage. The fraction of unentrapped BDP crystals was retained in the filter and was collected by dissolving using ethanol. From both, the entrapped and the unentrapped fractions, 0.2 mL of substrate was diluted with 0.8 mL of methanol and placed in a 2-mL vial. Due to the poor aqueous solubility of BDP (0.16 mg/ mL) (Sakagami et al., 2002), the amount of the drug dissolved in water during hydration was negligible, thus the calculation of drug entrapment efficiency was conducted considering that the drug was not soluble in water especially taking into account that the drug in all proliposome formulations constituted as high as 5 mole % of the lipid phase. For HPLC analysis, 20 mL of each sample was injected for analysis using an Agilent 1200HPLC system (Agilent Technologies, Germany) by injecting the sample through a C-18 column (Zorbax Eclipse XDB-C18, 4.6 mm 150 mm, 5 mm). Methanol and water (3:1 v/v) constituted the mobile phase and the flow rate of mobile phase and detection wavelength of the drug were set up at 1.7 mL/min and 239 nm respectively. 2.9. Statistical analysis All experiments were performed in quadruplets. Mean values SD and P-value (Student’s t-test unpaired, two-tail distribution)

was determined individually for all experiments with Microsoft Excel software. A P-value of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Scanning electron microscopy (SEM) SEM images of proliposomes prepared by coating the phospholipid onto sucrose carrier particles using the fluidized bed apparatus are shown in Fig. 2. Prior to coating with lipid, sucrose particles were apparently smooth and had flat edges without any depressions (Fig. 2a). By contrast, following coating with lipid, the resultant proliposome particles were generally granulated and had spherical depressions and textured surface (Fig. 2b, d and f). The drug-free proliposomes had spherical holes and textured surfaces at low coating level (5% weight gain, Fig. 2b), however, sucrose particles coated with a thicker lipid film (15% and 20% weight gain) showed a spherical bulges or impressions on the surface (Fig. 2d and f). Similar trend were noted in BDP loaded proliposomes (Fig. 2c, e and g). Due to the efficient drying mechanism of the fluidized bed dryer, ethanol was evaporated and lipids deposited on surface of the sucrose particles and formed a continuous film. The use of low temperature in the drying chamber ensured that a solid lipid film was formed on the sucrose particles with minimal particle agglomeration (Elhissi et al., 2012).

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Fig. 3. Size analysis of initial liposomes upon proliposome hydration. Error bars represent the standard deviation from the mean of four independent experiments.

Fig. 2. SEM images of (a) plain sucrose particle; (b), (c) and (d) 5%, 15% and 20% weight gain blank proliposome, and (e), (f) and (g) 5%, 15% and 20% weight gain BDP loaded proliposomes.

Owing to the small amount of drug compared to lipid and carrier, no apparent effect was seen on the proliposome particles when Fig. 2d and g were compared. It seems that the “bubbling” appearance on the surface of proliposomes happened only when lipid was coated with large concentration on sucrose, or when the coat contained lipid and BDP together. Further investigations are needed in the future using different drugs in order to investigate the role of drug on the surface morphology of proliposomes. 3.2. Size analysis of liposomes Size analysis of liposomes generated from proliposomes was performed before size reduction (Fig. 3). Particle size of liposomes was in the range of 5–7 mm with Span values being around 1.6. The incorporation of BDP in proliposomes did not significantly affect the size of the resultant liposomes for measurements conducted prior to size reduction. The obtained size range suggested that liposomes were large unilamellar vesicles (LUVs) or multilamellar vesicles (MLVs) (El-Ridy et al., 2007). However, our previous

investigations using sonicated BDP loaded soya phosphatidylcholine liposomes demonstrated that vesicles in this size range are predominantly small unilamellar as observed by electron microscopy (Elhissi et al., 2010). Size reduction was carried out using high pressure homogenization. The downsized vesicles were then freeze-dried followed by rehydration. The influence of size reduction and lyophilization on liposome size was assessed using dynamic light scattering (Table 1). For comparison purpose, the size reduction was carried out using probe sonication (Table 2). For 5%, 10% and 20% weight gain, liposomes produced with high pressure homogenizer were significantly smaller than those produced by probe sonication (P < 0.05). This indicates that the high pressure homogenizer generated higher shear forces compared to probe sonication at our experimental settings. These findings are in agreement with our recent work using niosomes (i.e., non-ionic surfactant vesicles) in which high pressure homogenization has shown to be more effective and efficient at reducing the size of vesicles compared to bath and probe sonication (Najlah et al., 2014). Small unilamellar vesicles are in the range of 25–100 nm (Lasic, 1988). The size of liposomes measured before freeze-drying was found to be in the range of 69.84–89.34 nm using high-pressure homogenization and 78.6–112.01 nm using probe-sonication. These findings indicate that high-pressure homogenization not only offered a possibility to generate nano-liposomes in larger quantities but also was more efficient at reducing the size of liposomes to the SUV range. After freeze-drying and rehydration, although the size of liposomes increased significantly for both set of formulations (p value