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The Depressurization of an Expanded Solution into Aqueous Media for the Bulk Production of Liposomes Louise A. Meure,† Robert Knott,‡ Neil R. Foster,† and Fariba Dehghani*,§ School of Chemical Sciences and Engineering, The UniVersity of New South Wales, Sydney, New South Wales 2052, Australia, Australian Nuclear Science and Technology Organisation, PriVate Mail Bag, Menai, New South Wales 2234, Australia, and School of Chemical and Biomolecular Engineering, The UniVersity of Sydney, Sydney, New South Wales 2006, Australia ReceiVed August 3, 2008. ReVised Manuscript ReceiVed October 27, 2008 A new dense gas process for the formation of liposomes has been developed: depressurization of an expanded solution into aqueous media (DESAM). The technique provides a fast and simple process for bulk liposome formation. As an alternative to current dense gas technologies, the DESAM process reduces the pressure requirements for liposome formation. Liposomes with diameters between 50 and 200 nm were formed. For all samples produced using ethanol as the solvent, the average effective diameter ranged from 119 to 207 nm. When chloroform was used as the solvent, the average effective diameter increased to 387 nm. The residual solvent volume fraction in the liposomal product was less than 4% v/v, which is approximately one-quarter of the value reported for some other dense gas liposome formation methods. The liposomal samples were stored after formation at 5 °C for up to 8 months, with the average effective diameter and polydispersity increasing by only 13% and 7%, respectively, indicating high stability of the formulations.
Introduction Liposomes form when phospholipids entropically self-assemble into vesicles in the presence of water, producing an aqueous medium surrounded by a lipid membrane. The lipids used to form liposomes are not water soluble and align in bilayer vesicles in order to minimize the unfavorable interactions between the bulk aqueous phase and the long hydrocarbon fatty acid chains.1 Liposomes (lipid vesicles) can be made up of a single bilayer, denoted as unilamellar vesicles, or multiple bilayers, referred to as multilamellar vesicles. Applications of Liposomes. Liposomes are ideal carriers for a variety of applications since a material can be entrapped either within the lipid membrane, such as for lipophilic/hydrophobic compounds, or within the aqueous interior, such as for hydrophilic compounds. Liposomes have been used as carriers for the delivery of dyes to textiles, genes and pesticides to plants, enzymes and nutritional supplements to foods, cosmetics to the skin, and pharmaceuticals to the body.2 However, liposomes are predominantly used for medical applications and can be used as drug carrier systems in order to increase the therapeutic index of drugs by decreasing drug toxicity, solubilizing drugs that have low aqueous solubility, and by protecting limited stability drugs from breakdown, which consequently increases their residence time in the body.3 Delivery of drugs using liposomes can also control the release of an incorporated drug, therefore improving bioavailability, as well as reduce the accumulation of drugs in sensitive tissue (such as heart, brain or kidneys) and target the drug to specific tissue.3 Liposomes can be produced from natural * Corresponding author. Tel: 612 9351 4794. Fax: 612 93512854. E-mail:
[email protected]. † The University of New South Wales. ‡ Australian Nuclear Science and Technology Organisation. § The University of Sydney. (1) New, R. C. C. Liposomes: A Practical Approach; Oxford University Press: New York, 1990. (2) Lasic, D., Barenholz, Y., Eds. Handbook of Nonmedical Applications of Liposomes: From Gene DeliVery and Diagnostics to Ecology; CRC Press: Boca Raton, FL, 1996. (3) Allen, T. M.; Moase, E. H. AdV. Drug DeliVery ReV. 1996, 21(2), 117–133.
materials so that the membrane bilayer is similar to the lipid portion of natural cell membranes and can thus be degraded by the same pathways.1 Natural purified phospholipids extracted from soybean, egg yolk, and sphingomyelin from egg or milk can be used for liposome formulation. Alternatively, liposomes can be produced entirely from artificial biocompatible components to provide improved chemical properties. Examples of synthetic phopsholipid derivatives that are used in commercial scale are phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylglycerol, phosphatidic, phosphatidylethanolamine, and phosphatidylserine. Production of Liposomes. Conventional liposome preparation methods have significant drawbacks since they are generally complicated, time-consuming, and are not easily scaleable for commercial production.4 The processes can also impose harsh conditions that can denature lipids or drugs, and there can be difficulties in achieving high encapsulation efficiencies. Toxic solvent residue in the product can be a significant disadvantage, as are the costs of handling toxic solvents and their residues. Dense gas processes have been developed for the production of liposomes in order to reduce the limitations involved in conventional preparation. The term dense gas is a general expression used to refer to a substance in the region surrounding the critical point. Dense gases possess properties intermediate to those of liquids and gases, with a liquid-like density and a diffusivity and viscosity between that of a liquid and a gas. Consequently, dense gases possess solvent power similar to that of liquids along with mass transport properties similar to those of gases. The use of dense gases, such as carbon dioxide (CO2), has enabled the organic solvent requirement to be reduced or eliminated compared to conventional production methods, which is particularly important for food and pharmaceutical applications. Dense gas processing can also provide sterile operating conditions and one-step production that can be easily transferred to large(4) Meure, L. A.; Foster, N. R.; Dehghani, F. AAPS PharmSciTech 2008, 9, 798–809.
10.1021/la802511a CCC: $40.75 2009 American Chemical Society Published on Web 12/10/2008
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scale operation.5,6 The most significant advantage of using dense gas technologies for the formation of liposomes is the dramatic reduction in processing time and the simple formation of bulk products. The major techniques described for applying dense gas technology to the formation of liposomes have been previously reviewed4 and include the injection method,7 decompression method,7 supercritical liposome method,5,8 and the supercritical reverse phase evaporation (scRPE) method.6 In the injection method, a compressed phase containing lipid, organic cosolvent, and compressed gas is sprayed into water, whereas in the decompression method the aqueous phase is incorporated into the compressed phase, which is sprayed into air. Castor and Chu claim their injection and decompression processes are capable of producing sterile, pharmaceutical-grade liposomes with a narrow particle size distribution that are substantially solvent free.9 Castor also reported the production of small uniform liposomes (or phospholipid nanosomes) using processes similar to the injection and decompression methods and referred to them as the SuperFluids phospholipid nanosome (SFS-CFN) manufacturing process.10 A number of variations on the operation of the process were investigated on the basis of the type of incorporated compound and its subsequent solubility in the aqueous or dense gas phase. The SFS-CFN process utilizes dense gases, polar cosolvents, and elevated temperatures and pressures to achieve solubilization of the raw materials for processing. The supercritical liposome method is a dense gas process similar to the injection method.5,8 The process involves the dissolution of phospholipid and cholesterol into supercritical CO2 at 60 °C and 250 bar using 5-7% v/v ethanol as a cosolvent. The lipid and cholesterol were dissolved in the CO2/ethanol phase after being placed in a cartridge through which repeated cycles of CO2/ethanol were passed. The solution was then rapidly expanded into an aqueous phase containing the hydrophilic compound to be entrapped. Bridson et al. also studied the production of liposomes using a method similar to the supercritical liposome method described by Frederiksen et al.11,12 The focus of the study was, however, to investigate the entrapment capabilities and the effects of process parameters on the product obtained. The authors concluded that liposomes with a broad particle size distribution were produced, and postformation processing may be required to improve the product. Otake et al. developed the scRPE method to produce liposomes using a dense gas.6 In the scRPE method, the lipid, organic cosolvent, and compressed gas were combined in a stirred, variable volume cell at 200 bar and 60 °C. An aqueous solution was then slowly introduced to the cell. The pressure was reduced by the release of the compressed gas, and liposomes were formed. Recently, Otake et al. developed an organic solvent free version of the scRPE process and referred to it as the improved (5) Anton, K.; Van Hoogevest, P.; Frederiksen, L. Preparation of a liposome dispersion containing an active agent by compression-decompression. European Patent 616801, 1994. (6) Otake, K.; Imura, T.; Sakai, H.; Abe, M. Langmuir 2001, 17(13), 3898– 3901. (7) Castor, T. P. Methods and apparatus for liposomes preparation. World Patent WO9427581, 1994. (8) Frederiksen, L.; Anton, K.; Barratt, B. J.; Van Hoogevest, P.; Leuenberger, H. 3rd International Symposium on Supercritical Fluids, Strasbourg, France, 1994. (9) Castor, T. P.; Chu, L. Methods and apparatus for making liposomes containing hydrophobic drugs. US Patent 5776486, 1998. (10) Castor, T. P. Curr. Drug DeliVery 2005, 2(4), 329–340. (11) Bridson, R. H.; Santos, R. C. D.; Al-Duri, B.; McAllister, S. M.; Robertson, J.; Alpar, H. O. J. Pharm. Pharmacol. 2006, 58(6), 775–785. (12) Alpar, H. O.; Santos, R. C. D.; Al-Duri, B.; McAllister, S. M.; Robertson, J.; Bridson, R. H. AAPS Annual Meeting and Exposition, Toronto, Canada , 2002.
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supercritical reverse phase evaporation (ISCRPE) method.13,14 In this method, an inhomogeneous mixture of phospholipid and aqueous solution was prepared using a stirrer, then the solution was pressurized by CO2 to 200 bar at 60 °C, and after 40 min the system was depressurized to generate liposomes. The drug loading efficiency and stability were improved in this method compared with the scRPE method because of the absence of ethanol in the processing and final formulation. Kadimi et al. recently developed a new method for preparing a drug-liposome formulation using compressed CO2.15 A solution of phospholipid, cholesterol, and amphotericin B-lipid complex in methanol-chloroform (1:1 volume ratio) was first prepared at 65 °C. Carbon dioxide that had been compressed to 150 bar was then used to expand the solution, using a magnetic stirrer to efficiently mix the components. After 20-30 min, a 0.9% saline solution was injected into the high pressure vessel at 60-65 °C, causing the spontaneous formation of liposomes. The compressed CO2 was released into saline solution to prevent the loss of any liposomal product. The liposomes produced were imaged using a phase contrast microscope, which showed particles ranging in size from 150 nm to 3 µm.15 The authors claimed the liposomes have a narrow particle size distribution, and they present a mean diameter of 4.5 µm with a standard deviation of 3.5 µm measured using a laser technique. The introduction of dense gas processing has enabled the rapid production of sterile liposomes of similar or improved quality compared to those produced by conventional methods without the use of toxic organic solvents. Despite the clear advantages to dense gas liposome production, there are still limitations since the dense gas processes described above generally require elevated pressures, commonly around 200-250 bar, and temperatures of 60 °C because of the low solubility of phospholipids in dense gases. Moreover, the processes do not provide a way for any organic solvent that is mixed with the dense gas to be removed from the system. Consequently, the volume fraction of residual solvent in the liposomal product can be quite high, which can dramatically reduce the stability of the liposomes. The aim of this study was to design and validate a new process for bulk liposome formation which utilizes the advantages of dense gases while overcoming the drawbacks of the current techniques. The new process operates at moderate temperatures and pressures below 60 bar, and the organic solvent used is washed from the system, leaving minimal residual solvent. The process developed is easily adaptable to a variety of incorporation compounds, including drug encapsulation and polymer for stabilization of the bilayer.
Experimental Section Materials. 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC with >99% purity) was purchased from Avanti Polar Lipids and was used as supplied. DSPC was used as an example phospholipid, while other types of phospholipids are equally applicable for use in the process. Cholesterol (99+ %) was purchased from Aldrich Chemical Co., Inc. Ethanol (99.7-100% v/v, BDH AnalaR, Merck Pty. Ltd.) and chloroform (99.8%, Spectrosol, Asia Pacific Specialty Chemicals, Ltd.) were used as solvents, and CO2 (99.5%, Linde Gas, Ltd.) was used as the dense gas. Various types of aqueous media were used, including water purified via reverse osmosis (RO) and deionized (DI) water from a Millipore Direct-Q 5 unit. TRIS buffered saline solution (10× concentrate, (13) Otake, K.; Shimomura, T.; Goto, T.; Imura, T.; Furuya, T.; Yoda, S.; Takebayashi, Y.; Sakai, H.; Abe, M. Langmuir 2006, 22(6), 2543–2550. (14) Otake, K.; Shimomura, T.; Goto, T.; Imura, T.; Furuya, T.; Yoda, S.; Takebayashi, Y.; Sakai, H.; Abe, M. Langmuir 2006, 22(9), 4054–4059. (15) Kadimi, U. S.; Balasubramanian, D. R.; Ganni, U. R.; Balaraman, M.; Govindarajulu, V. Nanomedicine 2007, 3(4), 273–280.
328 Langmuir, Vol. 25, No. 1, 2009 working solution containing 20 mM TRIS, pH approximately 7.4, and 0.9% NaCl) from Sigma Chemical Co. was also used. Deuterium oxide (D2O) (99.995 atom % isotopic purity) was obtained from Sigma Chemical Co. and used as received. Methanol (99.7% min., Unichrom, Ajax Finechem) and DI water (Millipore Direct-Q 5) were used during sample and mobile phase preparation for high-performance liquid chromatography (HPLC). Nitrogen (99.9%, Linde Gas Ltd.) was used in the evaporative light scattering detector (ELSD). Ethanol (99.7-100% v/v, BDH AnalaR, Merck Pty. Ltd.), methanol (99.7%, Unichrom, Asia Pacific Specialty Chemicals, Ltd.), toluene (Univar analytical reagent, AJAX Chemicals), chloroform (99.8%, Spectrosol, Asia Pacific Specialty Chemicals, Ltd.), ethyl acetate (99.9+ %, Burdick and Jackson), acetonitrile (99.7%, Unichrom, Asia Pacific Specialty Chemicals, Ltd.), DI water (Millipore Direct-Q 5), and Triton X-100 (t-octylphenoxy polyethoxyethanol, Sigma Chemical Co.) were used during sample preparation for gas chromatography (GC). Helium (g99.996%, Linde Gas, Ltd.) was used as the carrier gas, and compressed air (99.0%, Linde Gas, Ltd.) and hydrogen (99.99%, Linde Gas, Ltd.) were used in the flame ionization detector.
Procedure and Apparatus Phase Behavior Studies for Lipid-CO2-Solvent System. Melting Point Depression. A high pressure view cell (Thar Technologies, Inc., CL1521) was used to study the phase behavior of the lipid in CO2. The vessel used four heating cartridges, monitored by a thermocouple (RKC Instruments, Inc., CB100 digital controller) to control the temperature. The pressure of the system was monitored using a pressure transducer and indicator (Druck PDCR). Experiments were conducted at 50 and 70 °C to determine whether melting point depression occurs for DSPC exposed to CO2. The DSPC sample was held in front of the window of the high-pressure vessel using a sample holder. After thermal equilibrium was established at each desired temperature, the system was gradually pressurized. A syringe pump (ISCO Model 260D) was used to supply CO2 and control the pressure of the vessel. The pressure was increased at a slow and constant rate to 50 bar; the system was then left for 30 min to equilibrate. The pressure was then increased in 5 bar increments up to 350 bar at 50 °C and 150 bar at 70 °C, with a 10 min equilibration period between each increase in pressure. At each stage the physical state of the lipid was monitored visually. At the maximum pressure examined, the system was left for 2 h. Solubility Study. A dynamic solubility technique was used to assess the solubility of DSPC in high-pressure CO2 with/without cosolvent. The experimental apparatus and method was the same as that described previously by Charoenchaitrakool et al.16 Experiments were carried out at 50 °C in both pure CO2 at 250 and 280 bar, as well as in CO2-5 mol % ethanol at 250 bar. Ethanol as a cosolvent was supplied from an HPLC pump (Agilent 1100 Series) and homogeneously mixed with CO2 using a static mixer. A known quantity of dense gas passed through the high pressure vessel packed with lipid and the sample was collected at the exit upon depressurization, using a metering valve to control the flow rate and a filter to separate the extracted solid powder from CO2. A gravimetric technique was used to determine the mass of lipid dissolved in the known volume of CO2. At conditions where cosolvent was used; the sample collected was first dried to evaporate residual ethanol and then weighed. Threshold Pressure Measurement. The threshold pressure at which solute precipitates from a gas expanded solution was measured for lipid and cholesterol. Each solute was dissolved in an organic solvent and injected into a high pressure view cell (16) Charoenchaitrakool, M.; Dehghani, F.; Foster, N. R.; Chan, H. K. Ind. Eng. Chem. Res. 2000, 39(12), 4794–4802.
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Figure 1. Schematic diagram of the apparatus used for the DESAM process.17
(Jerguson sight gauge, series no. 32, 60 mL volume), which was placed within a thermally controlled water bath (Thermoline Australia Unistat 130 heater). The dense gas, CO2, was then supplied to the system from a syringe pump (ISCO 260D) to pressurize and expand the organic liquid. The pressure was monitored by a pressure indicator (Druck PDCR). Carbon dioxide was supplied to the bottom of the vessel, passing through a 0.5 µm frit, to enable efficient mixing with the organic liquid. The dense gas was gradually added to the vessel where it diffused into the organic liquid, expanded the solution, and led to supersaturation and precipitation of the solute. The pressure at which the precipitation occurred in the system was recorded as the threshold pressure. The threshold pressure was measured for precipitation of DSPC from ethanol and DSPC/cholesterol mixtures from ethanol and chloroform. For the DSPC solution, a solute concentration of 10 mg/mL was used at 22 °C. The lipid/cholesterol ethanol experiments were carried out using solute concentrations of 5, 10, and 20 mg/mL, with a lipid-to-cholesterol weight ratio of 70:30 and temperature of 22 °C. In each of these experiments, a slow pressurization rate of approximately 1 bar/min was used. The 5 mg/mL solute concentration was then repeated with a fast pressurization rate, where the pressure was increased to 50 bar in less than 1 min. In the chloroform experiments, a solute concentration of 20 mg/mL, a lipid-to-cholesterol weight ratio of 90:10, a temperature of 22 °C, and a moderate pressurization rate of 5 bar/min was used. The Depressurization of Expanded Solution into Aqueous Media (DESAM) Process for Liposome Formation. A schematic diagram of the apparatus for the DESAM process is shown in Figure 1.17 As shown in this figure, the major components of the DESAM apparatus are expansion and vesicle formation vessels. A solution comprising lipid and cholesterol in an organic solvent (ethanol or chloroform) was first injected via the injection port into the expansion chamber (Swagelok, 316 L-HDF4-150, 150 mL volume), which had a temperature between 20 and 23 °C. The solute concentration ranged from 5 to 20 mg solute/mL organic solvent, and lipid/cholesterol weight ratios of 90:10 and 70:30 were used. The pressure required for the expansion chamber was determined by the threshold pressure measurements. The vesicle formation chamber (Swagelok, 316 L-50DF4-150, 150 mL volume) was at ambient pressure and contained 50 mL of aqueous medium (RO water, DI water, or TRIS-buffered saline (17) Meure, L. A. The development of a novel process for the formation of liposomes: Depressurisation of an expanded solution into aqueous media (DESAM). Ph.D. Thesis, University of New South Wales, Sydney, Australia, 2004.
DESAM for Bulk Production of Liposomes
solution), then was placed in a paraffin oil bath heated to either 75 or 90 °C. In each run, CO2 was first sparged into the solution placed in the expansion chamber through the base of the vessel using a syringe pump (ISCO 260D). A pressure indicator (Druck PDCR) was used to monitor the pressure of the expansion chamber. The pressurization was carried out over 1-2 min, and approximately 10 min was allowed for the system to equilibrate once the expansion pressure was reached. At this stage, the line feeding dense gas to the base of the expansion chamber was closed and the valve at the top of the chamber opened. Carbon dioxide was then added to the expansion chamber from the top to keep the pressure constant. The expanded solution then passed from the pressurized expansion chamber into the ambient pressure vesicle formation chamber via a nozzle (0.9 cm length, 177.8 µm i.d. or 1.5 cm length, 254 µm i.d. stainless steel tubing) by opening the valve between the vessels. A filter (0.5 µm metal frit) in the bottom of the expansion chamber prevented any solutes that precipitated in the expansion chamber from passing to the vesicle formation chamber. Throughout spraying, CO2 was continuously fed to the expansion chamber to maintain the pressure of the system. Carbon dioxide (50-200 mL) was also passed through the system to assist the removal of organic solvent. The gas passed through the vesicle formation chamber to a solvent trap (Swagelok, 304 L-HDF4-75, 75 mL volume) that was immersed in a dry ice/acetone bath, then through a surge tank to collect any remaining solvent or solutes to a vent. The new technique is referred to as the DESAM process and is based on the dissolution of a lipid that is suitable for vesicle formation in a solvent, pressurization of the lipid solution through the addition of a dense gas to form an expanded lipid solution, and then the controlled release of the expanded lipid solution into heated aqueous media.17 The lipid solution pressurization and expansion is kept below the threshold pressure to avoid precipitation of the solutes. Controlled release of the expanded lipid solution is carried out while the pressure is maintained by further addition of dense gas. The gas and solvent leaving the system can be separated and recycled. The dense gas is used to expand the lipid solution through rapid diffusion of the gas into the solvent and assist in atomization upon depressurization. The fine droplets formed during depressurization assist the dispersion of lipid within the aqueous phase and improves the interaction between the components, aiding the formation of homogeneous liposomes. Moreover, the dense gas also functions as an agitation mechanism and a tool for solvent removal via entrainment with the gas. In conventional liposome formation techniques, agitation is often used either during or postformation to improve the size and homogeneity of the liposomes produced. The bubbling of the dense gas through the aqueous phase during the DESAM process can improve the characteristics of the liposomal product. Generally during liposome formation, aqueous solutions are heated above the lipid phase transition temperature, where the lipid exists in a liquidcrystal phase rather than a tightly ordered gel or solid phase, to improve the formation of liposomes. In the DESAM process, the elevated temperature in the vesicle formation chamber may also improve the removal of organic solvent from the product. The effects of DESAM process variables on the characteristics of liposomes produced were determined by varying nozzle diameter/flowrate, solute composition, solute concentration, organic solvent, vesicle formation chamber temperature, and volume of CO2 used for spraying. Each experimental condition was repeated at least three times, with the average result at each
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condition reported. The variation reported at each condition is the standard error. Product Characterization. Liposome Morphology. A transmission electron microscope (TEM) (Hitachi H-7000, 75 kV accelerating voltage) was used to assess the size and shape of the liposomes produced using the DESAM process. Samples were prepared for TEM analysis using negative staining. A drop of the liposomal suspension was placed onto a 200-mesh Formvarcoated copper grid, allowing approximately 1 min for the sample to adhere to the grid before the solution was drawn off using filter paper. A drop of 2% uranyl acetate aqueous solution was then placed on the grid and left for approximately 1 min before the grid was once again dried off using filter paper. The amount of stain applied to a sample has a dramatic affect on the types of structures observed. When a high level of stain was applied, the vesicles became separated, and any creases or crevices were more defined. When an even higher loading of stain was applied, the vesicles seemed to collapse and became shrivelled. Also, a stain gradient can occur across a grid if the drying off is not carried out effectively, producing regions stained to different extents on a single grid. The variety of observations obtained for liposomal samples correlate to the well-known unpredictability of negative staining.18,19 Consequently, great care was taken in preparing each sample in the same way, and multiple grids of each sample were prepared and examined. Liposome Structure. The liposomes were characterized to determine their internal structure using two small angle neutron scattering (SANS) facilities. The first was the 10m SANS instrument at the Australian Nuclear Science and Technology Organisation (ANSTO, Lucas Heights, Australia), and the second was the 30m SANS instrument at the NIST Centre for Neutron Research (NCNR) at the National Institute of Standards and Technology (NIST, Gaithersburg, MD). The ANSTO SANS instrument has a 5 m collimator and a 5 m sample-detector distance. The neutron wavelength, λ, was 3.5 Å with the wavelength spread, ∆λ/λ, approximately 15%. The neutron beam was 10 mm in diameter at the sample position, and the beamstop was 30 mm in diameter. Since the instrument is not located on a cold source, the neutron flux at 3.5 Å was limited to ∼3 × 105 cm-1 sec-1, and the q-range with this configuration was ∼0.01 < q < 0.15 Å-1 with the twodimensional (2D) detector in the 2θ ) 0 position (q is the scattering vector ) 4π sin θ/λ, and 2θ is the scattering angle). The data collection time for ∼10% counting statistics on the background was ∼2 h. In SANS, a beam of collimated radiation (neutron beam) is directed at a sample, and the radiation is scattered, absorbed, or transmitted by the sample. Neutrons are electrically neutral and pass through the electron cloud surrounding an atom and are scattered by the nucleus. A detector measures the scattering angle, 2θ, and intensity of the scattered radiation, I(q), across the sample, which provides the information about the size, shape, and orientation of the different components in the sample. SANS is able to differentiate between the various components on the basis of varying contrast, since the neutrons interact with the nucleus. For example, isotopes of hydrogen, which would appear to be the same in an X-ray beam, can be differentiated using SANS. Thus, water (H2O) and heavy water (D2O) are often combined in SANS to provide contrast with the sample and with each (18) Bugelski, P. J.; Sowinski, J. M.; Kirsh, R. L. In Liposomes: A Practical Approach; New, R. C. C., Ed.; Oxford University Press: New York, 1990; pp 140-154. (19) Hayat, M. A. Principles and Techniques of Electron Microscopy: Biological Applications; University Park Press: Baltimore, MD, 1981.
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other. In liposome studies, it can be useful to prepare liposomes in D2O to provide greater contrast with the lipid membrane. A liposome sample in D2O was first analyzed on the ANSTO SANS instrument less than 36 h after formation and again after storage for 1 week at 4 °C. The sample was prepared and loaded into quartz cells (with a path length of 5 mm) and maintained at 21.0 ( 0.5 °C during data collection. Since within experimental error there was no significant difference between the patterns collected at each time, the stability of the sample (at least in terms of characterization by SANS) was confirmed, and experiments on the high resolution SANS instrument at NIST in the USA could be planned. Another liposome sample was then prepared and transported on ice to the NCNR NG7 30 m SANS instrument at NIST. The elapsed time between sample preparation and the experiment was approximately one week. The instrument used pinhole collimation, and two instrument configurations were required to cover the planned q-range. For both configurations, the neutron wavelength was 8.09 Å, the sample aperture was 12.7 mm, and the beamstop size was 25.4 mm. In order to obtain a minimum q value of ∼0.0002 Å-1, the 2D detector was placed 15.3 m from the sample position, and focusing optics were inserted in the collimator. For the second configuration, the detector was moved to 1.5 m from the sample, and the source aperture increased from 14 mm, in the first configuration, to 50 mm. The maximum q value in the second configuration was ∼0.35 Å-1, which provided an excellent measure of the sample background. Data collection times for ∼1% counting statistics on the background were typically 60 min in the first configuration, and 30 min in the second configuration. The 2D SANS patterns for the liposome samples were corrected for background contributions and instrumental effects and then radially integrated with q ) 0 Å-1 as the center to produce onedimensional (1D) profiles. The profiles for the two instrument configurations were merged to provide a single profile that covered the range 0.0002 < q < 0.35 Å-1. The experimental data was fitted with a core/shell model and the scattering length density (SLD) determined for the core, the shell, and the bulk solvent.20 The SLD, F, of the sample components was calculated using the following equation. V is the average volume of a “component”, NA is Avogadro’s number (6.022 × 1023 mol-1), Fmass is the mass density, Mw is the molecular weight, and bi is the bound coherent scattering length of atom i. The bi is an intrinsic (measurable) property of a particular atom, and therefore the SLD for a group of atoms may be calculated if the Fmass is known or can be estimated. n
F)
∑ bi i
V¯
( )( ∑ )
) NA
Fmass Mw
bi
i
molecule
Liposomal Particle Size Distribution and Stability. Photon correlation spectroscopy (PCS) was used to assess the particle size distribution of the liposomal population using the Brookhaven ZetaPlus. Each liposomal sample was diluted in RO or DI water and placed in a disposable polypropylene cuvette. Ten runs, each of 1 min duration, were conducted at 23-25 °C for each sample. A laser wavelength of 678 nm was used with a destination angle of 90°. The dust cutoff was set between 20 and 50 µm. The instrument calculates an effective diameter for each run and an overall effective diameter for the 10 runs combined. The effective diameter is the mean diameter that is calculated by the following equation: (20) Kline, S. R. J. Appl. Crystallogr. 2006, 39(6), 895–900.
()
1 Effective diameter ) dk
-1
)
∑ Nidi6Pi i
∑ Nidi5Pi i
where Ni refers to the number per scattering volume of the ith particle, and Pi accounts for angular scattering effect for particles larger than λ/20. Pi is calculated using Mie theory and requires the particle refractive index; however, for Rayleigh scatters and at sufficiently low angles, Pi ) 1 is used in the program. Only particle size distribution data with an intensity weighting were considered in this study since the intensity weighted data is closest to the raw data collected and is the recommended form of use. The intensity weighting is placed on the diffusion coefficient, which varies inversely with particle diameter. The final effective diameter was calculated for each experimental condition by averaging the overall effective diameters from the repeat experiments conducted at each condition. The repeated analysis of a single sample over a period of time allowed the stability of the formulation to be examined. Liposome Composition. The liposome composition was characterized by developing a HPLC method to quantify the lipid and cholesterol content. A Waters HPLC system consisting of a 600 controller, 60F pump, in-line degasser, temperature control module, and 717 plus autosampler was used. The HPLC system was connected to a Polymer Laboratories ELSD (model: PL-ELS 1000). Nitrogen was used as the nebulizer gas in the ELSD with a gas flow of 1.2 SLM. The nebulizer temperature was set at 90 °C, and the evaporator temperature was set at 60 °C. An Agilent Zorbax Eclipse XDB-C8 column (4.6 × 150 mm2, 5 µm) was used. The mobile phase was 98% methanol and 2% DI water with a flowrate of 1 mL/min. The liposome samples were prepared for HPLC analysis by dissolution in methanol. Each sample was analyzed in triplicate with an injection volume of 10 µL and a run time of 15 min. Product Residual Solvent. The residual solvent in the liposomal product was quantified using GC. A Shimadzu gas chromatograph (GC-8A) with a flame ionization detector was used with a 6% cyanopropylphenyl polysiloxane, 94% dimethyl polysiloxane column (SGE, BPX624, 30 m length, 0.53 mm i.d., 3 µm film thickness). Helium was used as the carrier gas with a primary pressure of 7.5 kg/cm2. The column temperature was set at 100 °C throughout the analysis, and the injector/detector temperature was 230 °C. The preparation of the liposome samples for GC analysis involved the addition of a surfactant (Triton X-100) to lyse the vesicles. The solution was then filtered through Waters Sep-Pak Light cartridges with C18 sorbent to remove some of the lipid and cholesterol before injection. Filter tests were carried out to evaluate whether solvent may be absorbed by the filter, since this would alter the results obtained. It was found that, for ethanol, less than 1% v/v was retained by the filter. The effect of the filter was more significant for chloroform (16% v/v). Consequently, approximately two-thirds of a sample passed through the filter as a pretreatment during sample preparation and was then discarded as waste. The last portion was then passed through the filter and collected for analysis. An internal standard of methanol was also used in the GC analysis to account for discrepancies arising from injection volume and filtration effects. Each sample was injected in triplicate, using an injection volume of 0.1 µL. The run time for each sample was 10 min. Since chloroform is immiscible in water, a liquid extraction was carried out to determine the residual chloroform present. The volume of each of the liposomal samples was accurately measured, and then ethyl acetate was added to extract the chloroform. The
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Table 1. Summary of the Conditions Investigated and the Results Obtained for Producing Liposomes via the DESAM Processa set
1
2
3
4
5
6
7
8
9
nozzle diameter (µm) solute lipid content (% w/w) solute conc. (mg/mL) VFC temp. (°C ( 2.5) CO2 spraying vol. (mL) aqueous media organic solvent
254 70
178 70
178 90
178 90
178 90
178 90
178 90
178 90
178 90
20 75 200 RO H2O EtOH
20 75 200 RO H2O EtOH
20 75 200 RO H2O EtOH
5 75 200 RO H2O EtOH
20 90 200 RO H2O EtOH
20 75 50 RO H2O EtOH
20 75 200 DI H2O EtOH
20 75 200 TBS EtOH
20 75 200 RO H2O Chlfm
effective diameter (nm) polydispersity product lipid content (% w/w) residual solvent (% v/v)
156 ( 2 166 ( 5 121 ( 1 162 ( 23 207 ( 75 122 ( 2 119 ( 3 143 ( 5 387 ( 64 0.27 ( 0.01 0.27 ( 0.01 0.15 ( 0.01 0.19 ( 0.01 0.18 ( 0.01 0.15 ( 0.01 0.17 ( 0.02 0.18 ( 0.02 0.29 ( 0.03 75.2 ( 1.9 77.3 ( 1.8 82.4 ( 0.4 76.1 ( 0.1 81.8 ( 0.5 80.5 ( 0.2 80.9 ( 0.3 80.1 ( 0.6 81.8 ( 1.3 3.1 ( 0.4
1.6 ( 0.8
1.4 ( 0.4
2.2 ( 0.3
1.9 ( 0.7
3.9 ( 0.2
1.8 ( 0.4
2.0 ( 0.8
0.4 ( 0.3
a
VFC: vesicle formation chamber; RO H2O: water purified via reverse osmosis; DI H2O: deionized water; TBS: TRIS buffered saline; EtOH: ethanol; Chlfm: chloroform.
two-phase system was well mixed and left overnight to settle into two distinct phases. Samples were taken from each layer and analyzed for chloroform content.
Results and Discussion Phase Behavior Studies for Lipid-CO2-Solvent System. Prior to liposome formation, the phase behavior and solubility of the chosen lipid in dense CO2 were investigated to verify the suitability of the lipid for dense gas processing and, in particular, DESAM processing. Knowledge of the threshold pressure for precipitation of lipid from solution is also a key factor for design of the DESAM process in order to determine the maximum pressure for the technique, so that yield is enhanced and loss of lipid in the expansion chamber minimized. The solid state of DSPC was maintained when the lipid was exposed to CO2 below 350 bar at 50 °C and 150 bar at 70 °C. The solubility of DSPC in pure CO2 at 50 °C and pressures up to 280 bar was considered negligible. The addition of 5 mol % ethanol cosolvent did not significantly improve the solubility of DSPC in CO2 at 50 °C and 250 bar. Use of higher pressures or larger amounts of organic solvent are undesirable, thus the results of the solubility study are in agreement with the literature in concluding that effects arising from poor solubility of lipids in dense CO2 are not easily overcome. Frederiksen et al. found that, at 50 °C and 250 bar, DSPC required the addition of 4.8% v/v ethanol as well as the use of a recycling system for homogeneous dissolution of the lipid in CO2.21 The use of the DESAM process eliminates the current limitations of dense gas techniques associated with solubilizing lipids using a supercritical fluid and simply utilizes a dense gas as an aerosolization aid. The threshold pressure for the precipitation of DSPC from a 10 mg/mL ethanol solution at 22 °C was 55 bar. Precipitation was first observed at 58, 55, and 56 bar for the 5, 10, and 20 mg/mL solutions of DSPC and cholesterol (70:30 lipid to cholesterol weight ratio) in ethanol at 22 °C, respectively. Therefore it can be seen that cholesterol had negligible effect on the threshold pressure. When the pressurization rate for the 5 mg/mL lipid/cholesterol solution was dramatically increased, precipitation was not observed until 60 bar was reached. A faster pressurization rate is preferable for the DESAM process in order to minimize the time requirement for each experiment. During this experiment, noticeable expansion only started to occur after 50 bar was reached. Solution expansion is desired to maximize the effect of utilizing CO2 as an aerosolization aid to disperse (21) Frederiksen, L.; Anton, K.; van Hoogevest, P.; Keller, H. R.; Leuenberger, H. J. Pharm. Sci. 1997, 86(8), 921–928.
the lipid solutions throughout the aqueous phase. Therefore, the expansion pressure used in the DESAM experiments to avoid solute precipitation and enhance the yield for liposome formation from ethanol solutions was between 50 and 55 bar at 22 °C. The threshold pressure for the precipitation of a 20 mg/mL DSPC/cholesterol chloroform solution (90:10 lipid/cholesterol weight ratio) at 22 °C was 41 bar. The solvent volume had significantly expanded (doubled) by the time 40 bar was reached in the chloroform experiments. Therefore, expansion pressures between 38 and 40 bar were used for the DESAM chloroform experiments to achieve maximum expansion without lipid precipitation. Effects of Process Variables on DESAM Operation. The effects of solute composition, solute concentration, type of solvent, nozzle diameter, type of aqueous media, temperature of vesicle formation chamber, and volume of dense gas used for spraying on both the ease of operation of the DESAM process and the product were investigated. The results obtained for liposome formation are summarized in Table 1. Preliminary trials were conducted to establish viable nozzle options for the DESAM system. A variety of nozzles were tested including 102, 178, 254, 508, and 1016 µm i.d. stainless steel tubing and 100 µm i.d. Peeksil tubing (polymer tubing with fused silica lining). The most suitable nozzle for the DESAM apparatus, to control the flow rate and prevent blockages, was the 178 µm i.d. stainless steel tubing. The 254 µm nozzle was used in Set 1 (Table 1); however, there were difficulties in controlling the flow rate and maintaining constant pressure in the expansion chamber. Other nozzle dimensions may be selected depending on the pump capacity and vessel dimensions. The DESAM process is robust and, within the range examined, variation of solute concentration and composition, type of solvent, type of aqueous media, and volume of CO2 used for spraying had minimal effect on the operation of the DESAM process. The temperature of the vesicle formation chamber did, however, significantly affect the process since a smaller amount of liposomal product was obtained at 90 °C (Set 5) compared with 75 °C. The smaller volume can be attributed to the aqueous medium being closer to its boiling point at 90 °C, and thus some of the water was lost to the solvent trap via evaporation. Characterization of Liposomes Produced by the DESAM Process. Liposome Morphology. TEM was used to investigate the morphology of the particles produced in the DESAM process. At all conditions studied, submicron spheres were observed that possessed a similar structure to liposomes previously reported in the literature. The image shown in Figure 2 indicates that spherical particles, generally ranging in size from 35 to 200 nm
332 Langmuir, Vol. 25, No. 1, 2009
Figure 2. TEM image of liposomes produced in the DESAM process (Set 4).
Figure 3. The appearance of small spheres aggregating into larger spheres or captured within larger spheres in liposomal samples from the DESAM process (Set 7).
and more commonly 35-100 nm, were formed using the DESAM process. Images collected suggest that the liposomes were unilamellar. Not only were the spheres of a size range common to unilamellar liposomes, but in many images a single, thin wall can be seen at the edge of each particle. However, the arguments against positive identification of lamellarity using negative staining and TEM have been well documented in the literature.18 Staining artifacts are difficult to identify and are often interpreted as unexpected morphologies. Confirmation that the particles formed were in fact liposomes was found by utilizing SANS to identify an aqueous core, as discussed below. The spherical particles shown in Figure 2 are a general indication of the liposomes formed; however, some other features have also been observed. In several samples, a large quantity of smaller spherical particles (10-20 nm) was observed, which are at or below the lower size limit at which liposomes can be formed and may be considered as micelles. In some samples, small vesicles appear to be aggregated into or contained within a larger liposome vesicle, as shown in Figure 3. A vesicle-in-vesicle structure may be formed in the last stage of the DESAM process due to liposomes forming in the presence of existing vesicles. However, the lipid vesicles are more likely to have formed into aggregate structures during the negative staining process in order to minimize any deleterious effects when the aqueous phase was removed or to minimize the interactions of the lipid with the stain. The artifact of these aggregated systems could also result from a larger vesicle superimposed upon smaller vesicles, which is a common feature in TEM analysis. The particle size and morphology of the DESAM liposomes was not significantly changed within the range of process
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Figure 4. Rod or coffee-bean morphology observed in the liposomal samples produced by the DESAM process (Set 1).
parameters varied. However, rods or coffee bean morphology appeared in a few samples in addition to spherical particles, as shown in Figure 4. It is suggested that the coffee bean morphology was formed due to the collapse of vesicles, predominantly for the smaller particles. This effect can be attributed to the lower stability of small vesicles due to the high curvature of the membrane. A similar collapsed sphere feature has been observed by Johnson et al., who produced liposomal electron microscope images that are very similar to those found for the DESAM liposomes.22 The unilamellar liposomes produced by Johnson et al. using a conventional technique were stained with ammonium molybdate with and without the presence of protein. The images of the liposomes stained without protein showed “cup-like structures” and vesicles consisting of two lipid membranes. When protein was included in the staining process, the images showed vesicles consisting of a single lipid bilayer. Johnson et al. concluded that the liposomes in both images were unilamellar and that the vesicles had collapsed in the absence of protein. The double membrane feature can therefore be explained by the thick edge of the collapsed sphere, and the “cup-like structures” can be observed if the collapsed spheres are rotated. Collapsed spheres were present in all DESAM samples; however, rods or “coffee bean” particles were rare except in those samples from Sets 1 and 2, where a higher proportion of cholesterol was used compared with other samples. Cholesterol was incorporated in order to improve stability, and it has been reported in the literature that the incorporation of cholesterol causes larger liposomes to form.23 However, the rod-shaped particles were at the smaller end of the size range for the DESAM liposomes. Comparison of images from a number of samples indicated that the presence of rods may be promoted by the level of stain as well as the size of the vesicles. It is therefore also possible that the relative proportion of rods found in Sets 1 and 2 was amplified by the staining process. Because of the improved spherical morphology observed in Set 3, the remaining experiments were carried out using a lipid/cholesterol ratio of 90:10, thus enabling the product from Sets 4-9 to be compared to that of Set 3 as each parameter was altered. There was a high population of small particles (1 µm) particles in the PCS data. Since these large particles were not observed using microscopy and the existence of large particles is inconsistent, the very large particles are likely to be aggregates or agglomerates of smaller particles.
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Figure 5. Comparison between the multimodal size distribution for (a) ethanol (Set 3) and (b) chloroform (Set 9) DESAM samples.
The liposome effective diameter increased when the temperature of the vesicle formation chamber was increased from 75 to 90 °C (Set 5), due to a proportion of particles greater than 4 µm being detected. These large particles are also likely to be fused or aggregated from smaller particles. At 90 °C, the vesicle formation chamber temperature was significantly above the lipid phase transition temperature (55 °C), increasing lipid fluidity and the chance of vesicle fusion. In addition, a significant quantity of water evaporated at 90 °C and was thus removed from the system, changing the formation environment. The average polydispersity of the DESAM liposome samples ranged from 0.15 to 0.29, as shown in Table 1. The polydispersity reported by the PCS instrument is the relative variance and is a measure of the width of the size distribution. The most polydisperse samples (0.29) were from Set 9, when chloroform was used; however, the ethanol samples still ranged from 0.15 to 0.27. It is interesting to notice that the two experimental conditions where lower solute lipid content was used (Sets 1 and 2) both showed a high polydispersity of 0.27, and these were the samples containing a large proportion of rod or “coffee bean” morphologies. All of the ethanol samples produced using a solute lipid content of approximately 90 wt% produced a polydispersity between 0.15 and 0.20. Despite the differences caused by solvent type or lipid/cholesterol mass ratio, all the samples were similarly and acceptably polydisperse. Generally, the particle size of liposomes produced at each condition was highly reproducible. However, it can be seen that the standard error was high for three sets of experiments: Set 4 with lower solute concentration (162 ( 23 nm); Set 5 with higher vesicle formation chamber temperature (207 ( 75 nm); and Set 9 with chloroform solutions (387 ( 64 nm). It is believed that the larger error is due to varying levels of particle aggregation, with some samples at these conditions containing aggregates while others did not. Analysis of the samples using TEM did not show any particles larger than 1 µm, or even close to 1 µm in
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Figure 6. Multimodal size distribution for the liposomal sample produced in Set 7, analyzed using the Brookhaven ZetaPlus (a) initially and (b) after five months.
diameter. The TEM study therefore suggests that the PCS results provide artificially large variations between samples and polydispersity readings due to particle aggregation. In this study, the liposomal size has been evaluated using microscopy, PCS, and SANS. All particle sizing equipment use certain assumptions to provide the distribution data. A single DESAM sample was analyzed on a different PCS instrument, the Malvern Instruments Zetasizer Nano ZS, based on the same principles as the Brookhaven ZetaPlus. A sample from Set 7 was selected for this comparison, with the average size found to be 104 nm and the polydispersity was 0.11. The two Brookhaven results for the sample (taken at a similar time as the Zetasizer and again 5 months later) were effective diameters of 111 and 121 nm and polydispersities of 0.16 and 0.19. The multimodal size distributions attained using the Brookhaven for the Set 7 sample are given in Figure 6. Interestingly, it seems that larger particles (greater than 1 µm) were only found in the sample when it was analyzed soon after formation. These results reinforce the hypothesis that aggregates of primary particles are produced during the PCS analysis and suggest that the aggregates can be redispersed as individual particles. The results imply that the varying lengths of time between sample preparation and analysis are not of great concern, and the stability of the formulation is high. The size distribution obtained within a few weeks of each other using the two instruments are extremely similar and confirm the result acquired. Liposomes produced by other dense gas techniques have also been characterized by PCS. Castor and Chu prepared liposomes using both the decompression and injection methods and characterized the particle size distribution using PCS.7,9,25 Smaller vesicles were able to be produced using the decompression method than with the injection method. The difference between the two (25) Castor, T. P.; Chu, L. Methods and apparatus for making liposomes containing hydrophobic drugs. World Patent WO9615774, 1996.
DESAM for Bulk Production of Liposomes
processes can be attributed to the different particle formation mechanisms, and the DESAM process is expected to have a similar particle formation mechanism to that of the injection method. The average sizes of the liposomes produced in the injection method were 478 and 326 nm when 500 and 60 µm nozzles were used, respectively.25 The standard deviation of these samples was 150-180 nm. Castor and Chu investigated the effect of the pressure of the dense gas phase prior to depressurization on the size of the liposomes produced. It was found that, at higher pressures of 207 bar, larger liposomes were produced. The formation of liposomes was also tested by Castor and Chu with and without the presence of a variety of cosolvents.25 The effect of cosolvent was investigated using nitrous oxide, propane, and a refrigerant as the dense gas, but not with CO2. It was found that the use of cosolvent prevented the formation of micronsized particles. Polar cosolvents reduce the surface tension of water and subsequently the interfacial tension between the dense gas and aqueous phase.25 The lower interfacial tension may affect the size of the bubbles formed and thus the liposome size. The liposomes produced in the DESAM process are smaller than those produced in the injection method, despite the similar particle formation mechanism. The difference may be attributed to the lower pressure used and the higher proportion of ethanol that comes into contact with the aqueous phase as the lipid precipitates in the DESAM process. Frederiksen et al. reported that the liposomes produced in the supercritical liposome method had an average size of 200 nm, based on PCS measurements, with a polydispersity of 0.6 and a standard deviation of 25 nm.21 Frederiksen et al. reported that the size distribution is affected by the internal diameter of the encapsulation capillary.8 Using a capillary with an internal diameter greater than 500 µm, a polydisperse sample containing 1 µm vesicles was formed, whereas 40-50 nm liposomes were formed with a smaller diameter capillary. The liposomes produced by Bridson et al. using essentially the same technique as the supercritical liposome method produced a broad particle size distribution that spanned from approximately 50 nm to greater than 1 µm, with a polydispersity between 0.5 and 1.0.11 The average particle size of these samples ranged from 105 nm to 2 µm, and Bridson et al. speculated that, based on their investigations, the samples produced by Frederiksen et al. were likely to have also contained larger vesicles as well as the smaller vesicles reported. Bridson et al. observed that significantly greater polydispersity and larger particles were produced when samples were processed using pressures below 250 bar, and at 150 bar there was a negligible amount of submicron particles.11 The results imply that the use of high pressures in the supercritical liposome method may be unavoidable for the production of small vesicles, since at low pressures the phospholipid cannot be solubilized in the dense gas phase. Interestingly, the smallest average particle size was obtained for samples without cholesterol present. Bridson et al. attribute this trend to the presence of cholesterol increasing vesicle aggregation and fusion, rather than to the actual formation of smaller individual vesicles.11 The use of a smaller proportion of cholesterol (10 w/w % as compared to 30 w/w %) in the DESAM process produced a slight reduction in the mean diameter from 166 to 121 nm. The PCS results obtained for the DESAM process correlate well with the results obtained in the supercritical liposome method, with a large number of small particles and some larger particles. The polydispersity of the DESAM samples is significantly lower, and it is believed that the larger particles found using PCS analysis were in fact aggregates of smaller vesicles, as discussed above, particularly due to the absence of
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any micron sized vesicles when analyzing the samples using microscopy. In contrast to this, Bridson et al. suggest that, although aggregation cannot be dismissed in their process, since micronsized particles were observed using microscopy, then it is likely that nano- and micron-sized populations were both present in most samples.11 The particle size distributions presented by Imura et al. for the scRPE method26 are also similar to those found in the DESAM study. When pressures below 200 bar were used, smaller liposomes with a bimodal size distribution were formed with a small peak between 200-400 nm and a large peak at approximately 1 µm. Above 200 bar, larger liposomes with a broad monomodal distribution spanning from approximately 100 nm to 1 µm were found. Imura et al. showed that the particle size was dependent upon both the pressure of the system and the presence of ethanol.26 In the ISCRPE process, the size of the liposomes was controlled by the rate of depressurisation, with slower depressurization rates producing larger liposomes.13 It is clearly evident that the liposomes produced in the DESAM process have an average size that is similar to or smaller than those produced by other dense gas techniques. Lower pressures and moderate depressurization rates were also able to be used to produce liposomal populations with decreased polydispersity. Liposome Composition. The composition of the liposomal product formed using the DESAM process was characterized using HPLC. The concentration of cholesterol and lipid in each sample was determined, and then the ratio of lipid to cholesterol was calculated. The concentration values determined for lipid and cholesterol were converted to liposomal membrane composition, assuming the membrane was made up entirely of DSPC and cholesterol and assuming that all lipids and cholesterol present form into liposomes. Under all experimental conditions, the liposomal membrane possessed a lipid composition between 75 and 82% w/w (shown in Table 1), with the remainder being made up of cholesterol. Since solute lipid compositions prior to processing of 70% w/w and 90% w/w were used, this is an intriguing result. It appears that, regardless of whether the initial lipid composition is 70 or 90% w/w, the liposome membrane lipid composition is close to 80% w/w. Variation of experimental conditions had minimal effects on the lipid content of the liposomes produced. The reproducibility between the lipid content in different samples produced using the same experimental conditions was high since the largest deviation from the mean in any sample set was 5%. The data collected also indicated high yield of lipid and cholesterol in liposome formulation using the DESAM process. For Sets 1 and 2, the lipid composition in the liposome membrane was 75-77% w/w, while the preprocessing content was 70% w/w. The increase in the lipid composition during processing could be attributed to loss of cholesterol through slight precipitation of cholesterol in the expansion chamber or slight dissolution of cholesterol in CO2. It may be possible that some of the cholesterol was entrained with the CO2 and removed from the system. For the experimental sets conducted using a solute lipid content of 90% w/w, the average lipid composition in the liposome membrane was consistent and generally ranged from 80-82% w/w. The decreased lipid composition is not unexpected and can be attributed to lipid loss or lipid degradation. Phospholipids are known for undergoing hydrolytic degradation, particularly during storage over time. Only DSPC was examined during the HPLC analysis, and any lipid degradation products (26) Imura, T.; Gotoh, T.; Otake, K.; Yoda, S.; Takebayashi, Y.; Yokoyama, S.; Takebayashi, H.; Sakai, H.; Yuasa, M.; Abe, M. Langmuir 2003, 19(6), 2021– 2025.
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were not quantified. Therefore, if some of the DSPC had been changed into another form during storage, it would not have been quantified, and thus the lipid content would seem to be less. An intriguing observation is that the decreased lipid composition remained consistent between samples. It can therefore be concluded that lipid degradation was not a significant effect for the DESAM samples since lipid degradation would affect each sample differently depending on the time since formation. Since there is exceptional consistency between samples, it must be concluded that there are secondary factors causing the decreased lipid-to-cholesterol ratio. The fact that the liposomes formed had a cholesterol composition between 18 and 25% w/w demonstrates the ability of the DESAM process to be used to produce liposomes encapsulating hydrophobic materials. The inclusion of hydrophobic materials into liposomal formulations is carried out by incorporating them into the lipid membrane. Otake et al. investigated the trapping efficiency of liposomes produced using the scRPE method for hydrophobic substances by incorporating cholesterol and found that 63% of the initial cholesterol was present in the final formulation.6 Otake et al. then concluded that liposomes with a high trapping efficiency for oil-soluble substances can be produced using the scRPE method. Similarly, the results obtained in this study validate the use of the DESAM process for producing liposomes incorporating hydrophobic materials. Product Residual SolVent. The average residual solvent level in the liposomes produced using the DESAM process under all conditions investigated was less than 4% v/v. At each condition for which ethanol was used as a solvent, the average residual solvent level ranged from 1.4 to 3.9% v/v. When chloroform was used as a solvent (Set 9), the average residual solvent concentration was 0.4% v/v. The lower residual solvent concentration attained when chloroform was used as the solvent was anticipated since chloroform is immiscible with water. Therefore, it is expected that chloroform would be more easily removed from the aqueous phase during washing. The average of the total chloroform concentration for the products from Set 9 was 0.4% v/v; however, the residual chloroform found in all samples was minimal at less than 1% v/v and may be further reduced through manipulation of the experimental parameters. There were two experimental parameters that were varied predominantly to evaluate their effect on product residual solvent levels: the temperature used for the vesicle formation chamber, and the volume of CO2 used for spraying. It was hypothesized that an increased vesicle formation chamber temperature would reduce the amount of solvent that remained in the product. However, no noticeable effect was observed. The vesicle formation chamber temperature must remain above the lipid phase transition temperature (55 °C) and below the aqueous phase boiling point (100 °C), thus 90 °C is considered to be the highest suitable working temperature. The volume of CO2 used for spraying did, however, have a significant impact on the residual solvent. The residual solvent was decreased from 3.9% v/v to 1.4% v/v simply by increasing the amount of CO2 used for spraying from 50 to 200 mL at processing pressure. Consequently, an even larger volume of dense gas could be passed through the sample in order to further reduce the organic solvent concentrations. The residual solvent concentrations found using the DESAM process are dramatically lower than those of some other dense gas liposome formation techniques that utilize organic solvents, such as the supercritical liposome method with 14-17% v/v residual solvent.21
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Advantages of the DESAM Process for Bulk Liposome Formation. The DESAM process has many advantages over conventional liposome formation techniques. These advantages include the fact that it is a simple and rapid process for bulk production of unilamellar liposomes. A conventional liposome standard was produced, and the formation process took almost 24 h and multiple stages to complete. The DESAM process produced a greater volume of the same formulation in less than half an hour, clearly demonstrating the dramatic reduction in processing time. The conventional ethanol27 and ether28,29 injection methods exhibit some similarities to the DESAM process since they involve the dissolution of a lipid into an organic phase, followed by the injection of the lipid solution into aqueous media forming liposomes. The potential drawbacks of the ethanol injection method are the poor homogeneity of the vesicles if there is not adequate mixing and the residual solvent levels in the product. The ether injection method eliminates the residual solvent issue by having a heated aqueous phase, but is a timeconsuming technique. It has been suggested that injecting the ether solution at a rate faster than 0.2 mL/min can cause cooling of the aqueous phase due to evaporation, and that pre-evaporation of ether can cause nozzle blockages and the formation of multilamellar vesicles.30 The DESAM process has significant advantages over both the ethanol and ether injection methods since the depressurization from a high pressure environment creates outstanding dispersion of the lipid solution and mixing with the aqueous environment. The incorporation of both heating and dense gas washing enables the solvent to be efficiently removed. The DESAM process can also produce an equivalent volume of product in a significantly reduced time span. Compared with other dense gas processes developed for liposome formation, the DESAM process is beneficial due to its simplicity and the incorporation of residual solvent removal measures into the method. The DESAM process also operates at pressures generally less than 60 bar and moderate temperatures, therefore making the process more cost-effective and avoiding the concerns of uncontrollable foam formation present in the supercritical liposome method. A significant advantage of the DESAM process is that it can be used to process a broad range of materials since there is no requirement for the compound to be solubilized in the dense gas and there are no high shear forces. Furthermore, time-consuming solubility studies and recycling loops for lipid solubilization are not needed. The only preliminary investigation required is the determination of the threshold pressure for precipitation of the solutes from expanded solution, such that the solution expansion can be carried out without precipitation. In the DESAM process, the entrapment of hydrophilic compounds may be achieved through the dissolution of the target compound into the aqueous media prior to release of the lipid solution. The liposomes would then form, entrapping the hydrophilic compound within the aqueous interior of the vesicle. To entrap a hydrophobic, lipophilic, or amphipathic compound into liposomes using the DESAM process, the compound is dissolved along with the phospholipid and other solutes in the liquid solvent. It is believed that the compound then becomes entrapped within the phospholipid membrane as a result of the affinity of the compound for the membrane rather than the aqueous phase. The suitability of the DESAM process for entrapping (27) Batzri, S.; Korn, E. D. Biochim. Biophys. Acta 1973, 298(4), 1015–1019. (28) Deamer, D.; Bangham, A. D. Biochim. Biophys. Acta 1976, 443(3), 629– 634. (29) Deamer, D. W. Ann. N.Y. Acad. Sci. 1978, 308, 250–258. (30) New R. C. C. In Liposomes: A Practical Approach; New, R. C. C., Ed.; Oxford University Press: New York, 1990; pp 33-104.
DESAM for Bulk Production of Liposomes
hydrophobic compounds has already been demonstrated through incorporating up to 25% w/w cholesterol into the liposome formulation. The DESAM technique can also be applied to the formation of structures other than liposomes. Microparticles of hydrophobic compounds could be produced through precipitation into aqueous media in the DESAM process.
Conclusions The DESAM process has been used to produce unilamellar liposomes that generally range in size from 50 to 200 nm at moderate temperatures and pressures. The average effective diameter of the liposomal populations ranged from 119 to 387 nm. The process was robust and reproducible in the production of vesicles in this size range over a broad range of experimental conditions, with the polydispersity remaining below 0.29. Confirmation of liposome formation has been achieved through identification of an aqueous core in the particles by SANS. The residual solvent volume fraction in the liposomal product was
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less than 4% v/v, which is significantly less than that reported for other dense gas liposome formation techniques. The DESAM process is a fast and simple bulk process capable of producing stable liposomal vesicles without the requirement for extreme conditions. There is great potential for the DESAM process to be further investigated for the production of liposomal formulations, including the incorporation of polymer as well as hydrophilic and hydrophobic compounds, which is the focus of our current studies. Acknowledgment. The authors gratefully acknowledge the financial support of the Australian CRC for Polymers. We acknowledge the support of the National Institute of Standards and Technology, U.S. Department of Commerce, in providing the neutron research facilities used in this work. This work utilized facilities supported in part by the National Science Foundation under Agreement No. DMR-9986442. LA802511A
