Recent Applications of Supercritical Fluid Technology to Pharmaceutical Powder Systemst

The unique characteristics of supercritical fluids can be utilised to overcome the drawbacks associated with the conventional techniques for the generation of pharmaceutical powders. These characteristics include high diffusivity compared with those of liquids, relatively high solvent density and solvent power intermediate between those of gases and liquids. Such characteristics make it possible to use supercritical fluids either as solvents or antisolvents. The application of supercritical fluid technology to various pharmaceutical processes such as micronisation of a wide spectrum of pharmaceutical compounds, encapsulation and polymer impregnation, coprecipitation of Pharmaceutical compounds and liposome formation, has been explored. The present review critically discusses the novel supercritical fluid techniques that are currently available and the applications of such techniques to pharmaceutical powder systems.


Introduction
Supercritical fluid technology includes a broad range of processes that utilise the properties of fluids at operating conditions above both the critical temperature and pressure of the solvent.The technology offers tremendous potential for the pharmaceutical and food industries, forest product industries, separations and chemical processing, and for the processing of polymers.
The use of supercritical fluids (SCF) is a relatively new approach in pharmaceutical research.The low critical temperature and pressure of SCFs such as carbon dioxide (C0 2 ) (Tc=31.1 °C, Pc=72.9 atm) make them attractive for use in the processing of pharmaceuticals.Supercritical fluid technology provides viable alternatives to conventional methods of size reduction and has significant potential in the preparation of fine powders of thermally labile drugs.In particular, the technology offers a solvent-free process for the preparation of drug-loaded microspheres.Conventional techniques for microencapsulation of pharmaceuticals, such as double emulsion, utilise large amounts of organic solvents.Health concerns associated with solvent emissions from conventional pharmaceutical processes, difficulties associated with the removal of residual solvent from the product, and the drive for energy efficient and inexpensive processes have provided an incentive for increased focus on the development of novel clean technologies.Supercritical fluid processes are generally environmentally acceptable, and often require fewer processing steps than conventional methods.
Supercritical fluids can be used for recrystallisation as solvents as well as antisolvents.In the antisolvent mode, a SCF can be added to a solution, or vice-versa, to induce precipitation of the solid of interest.As with traditional solvents, solutions of supercritical fluids can also be used in spray-drying processes.
Certain limitations need to be addressed before the full potential of supercritical fluid technology can be realised.Among the challenges facing successful implementation of the technology is the lack of complete understanding of the fundamental principles involved and the costs associated with the specialised equipment required.
Supercritical fluid technology has been successfully employed at laboratory and pilot plant scales, and the pharmaceutical industry is beginning to exploit the technology commercially.This review covers recent applications of supercritical fluid technology for processing of pharmaceutical powder systems.

Fundamentals
Each compound possesses a critical temperature (Tc) and pressure (Pc) above which no liquid phase formation can be induced by further compression.A pressure-temperature phase diagram of a pure component is shown in Figure 1.The lines divide the P-T diagram into three regions: solid, gas, and liquid.The three phase boundary lines meet at the triple point at which all phases are in equilibrium.As the pressure and temperature increase, the critical point will eventually be reached.At this condition the gas and liquid have the same density, and exist as a single phase.The compressibility of a compound approaches infinity as the critical point is approached.In the vicinity of the critical region, the thermal-physical properties exhibit very high rates of change with respect to temperature and pressure.Along a near-critical isotherm (between Tc=l.O and 1.2), the density and transport properties, such as viscosity and diffusivity, as well as other physical properties, such as dielectric constant   and solvent strength, can be varied continuously from gas-like to liquid-like with relatively small changes around the critical pressure (0.9-2.0 Pc).Supercritical fluids possess solvent density and solvent powers intermediate to those of liquids and gases.Solute solubilities in SCFs can be in the range of 3 to 12 orders of magnitude higher than those predicted by ideal gas behaviour.Supercritical fluids have solute molecular diffusivities much higher than liquids and viscosities intermediate to those of gases and liquids.Supercritical fluid properties can be easily controlled between liquid-like and gas-like extremes by changing the pressure.
The solvents that are commonly used as supercritical fluids are listed in

Supercritical Fluid Techniques in Powder Generation
The first observation of changes in particle size and morphology upon the expansion of supercritical solutions was made by Hannay and Hogarth [2], who characterised precipitated solids as snow in a gas and frost on glass.More than a century elapsed before Smith et al. (1986)  [3] carried out the first investigations of rapid expansion of supercritical solutions, demonstrating the potential of this technique for particle formation, size reduction, and comminution of a wide variety of materials.
Supercritical fluids as solvents or antisolvents can be used for the generation of fine powders.The process is called rapid expansion of supercritical solutions (RESS) when the SCF is a solvent and is used when the compound of interest is soluble in the supercritical fluid.The technology is known as the gas antisolvent (GAS) process, supercritical antisolvent (SAS) process, aerosol solvent extraction system (ASES) or solution-enhanced dispersion by supercritical fluids (SEDS) when the SCF is an antisolvent.These techniques are used when the compound has limited solubility in the SCF.Two other areas in which dense gases and SCFs are utilised for powder generation are supercritical fluid drug delivery (SFDD) and impregnation of polymers with pharmaceutical compounds.

Rapid Expansion of Supercritical Fluids
The rapid expansion of supercritical solutions is a novel method of particle formation, recrystallisation and particle size reduction, formation of thin films, microencapsulation, and powder mixing [4][5][6].In this process, the solute is first dissolved in a supercritical fluid, which is then subjected to rapid expansion by passing through a nozzle at sonic speeds.During expansion, the density and solubilizing power of the supercritical fluid decreases dramatically, resulting in a high degree of solute supersaturation and subsequent precipitation of solute particles [7].The combination of high supersaturation ratios and a rapid propagating mechanical perturbation is a distinguishing characteristic of the RESS process [8].
A schematic diagram of a typical RESS set up is shown in Figure 2. In general the RESS apparatus consists of two main units: the extraction or solubilization unit and the precipitation unit.In the extraction unit, the extraction vessel is packed with the solute and placed in a constant temperature environment.The supercritical fluid is then passed through a preheater and the vessel at a particular temperature and pressure.After equilibrium, the saturated solution is passed through an expansion device, such as a capillary nozzle [8,9], laser drilled nozzle [5,8,9] or frit nozzle [10], which is located in the precipitation unit.The temperature-controlled nozzle is used to define the pre-expansion temperature.Heating of the nozzle and the connections between the extraction and the precipitation units is necessary to prevent condensation and phase changes and premature precipitation.The temperature is typically higher than the extraction temperature.During expansion or decompression, the density and solubilising power of the supercritical fluid decreases dramatically, resulting in a high degree of solute supersaturation and subsequent precipitation.
The major limitation of using RESS in pharmaceutical applications is that some pharmaceutical compounds including high molecular weight compounds, such as proteins and polymers, are insoluble in supercritical carbon dioxide at moderate conditions ( <60°C and 300 bar).The low solubility necessitates either using large quantities of the supercritical fluid, or using polar co-solvents, which can complicate the phase behaviour of the system and may remain in the precipitated product.In addition, the scale-up of the process requires a comprehensive understanding of the underlying physical phenomena and the development of mathematical models to predict the characteristics of the product for a given set of operating conditions.

Supercritical Antisolvent Techniques
The low solubility of the pharmaceutical compounds in SCFs can be used to advantage by employing a dense gas (fluids close to the critical point) as anti-solvents.In the gas antisolvent techniques, the solute is soluble in the organic solvent, which in turn must be completely or partially miscible with the dense gas.There are two modes of performing gas antisolvent recrystallisation.The first mode, known simply as the GAS process, involves the gradual addition of anti-solvent to the organic solution containing the solute until precipitation occurs [11].The second mode, known as the ASES, involves introducing the organic solution of the solute through a capillary nozzle into a flowing dense gas stream [12].The recent high level of interest in antisolvent techniques stems from the fact that they are more flexible than conventional processes and are broadly applicable.

GAS
It is possible to induce rapid crystallisation by introducing the antisolvent gas into a solution containing dissolved solute.The dissolution of the dense gas in liquids is often accompanied by large volume expansion, and consequently a reduction in the solvent power of the liquid.Rapid addition of SCF results in a sudden reduction in the density of the liquid, a drastic rise in the supersaturation within the liquid mixture, and the subsequent formation of small and uniform particles.The process is a semi-batch and is known as gas antisolvent recrystallisation [11,13].A schematic diagram of a typical GAS set-up is presented in Figure 3.The standard GAS procedure begins by passing the antisolvent through a pre-heater into the precipitation chamber in which an organic solvent containing the solute has been charged.The homogeneity of the gas and the liquid during expansion is maintained by mechanical agitation, or by using a frit as a gas sparger.Once particle formation or crystallisation is complete, the antisolvent is delivered at a constant pressure to the vessel to wash the precipitate, and the system is then depressurised followed by powder collection.
Many of the experimental designs rely on the gas bubbling through the solution for mixing.This method of mixing has been proved to be as efficient as a magnetic stirrer for small volume crystallisation vessel [14].In larger vessels, such as those exceeding one-litre, an efficient mixer is required to keep a uniform concentration in the expanded solution.
Process parameters that may influence particle formation in GAS precipitation include the rate of expansion of the solution, the physico-chemical properties of the solvent and antisolvent type [15,16], the concentration of the solute [15, 17, 18], temperature [17,18], and agitation and stirring [14].
Precipitation chamber Fig. 3 A schematic diagram of the gas antisolvent apparatus.

ASES
In addition to the ASES, other terms for the continuous gas anti-solvent process have been reported in the literature.The various techniques are similar and include supercritical antisolvent (SAS), precipitation with compressed antisolvents (PCA), and the solution enhanced dispersion by supercritical fluids (SEDS).
In the ASES process, the solute can be precipitated by spraying the liquid solution into a dense gas or supercritical fluid.The process is continuous compared to the GAS process, the latter being a batch or semi-batch process.The basic experimental set-up is shown in Figure 4.The liquid containing the solute and the dense gas are fed continuously into the precipitation chamber.The liquid solution is aerosolized through a small orifice or capillary nozzle (i.d.20-500 !J.m) and the droplets come in contact with the antisolvent.The antisolvent rapidly diffuses into the liquid solvent as the carrier liquid solvent diffuses into the antisolvent.The expanded solvent has a lower solvent strength than the pure solvent; thus the mixture becomes supersaturated resulting in the precipitation of the solute [12,19].After precipitation ofthe solute, the solid is filtered and washed from traces of solvent by the addition of antisolvent.
The basic experimental apparatus has been modified to tailor the process to specific applications.For micronisation applications, nozzles of different diameters and designs have been employed to control particle size and distribution, and agglomeration of precipitated products.Nozzles and orifice sizes employed in the studies to date have ranged from 20 11m to 500 IJ.m.Ultrasonic [20], hi-energy [21], double [22,23] and triple coaxial nozzle arrangements have been incorporated to further improve the quality of micro- nised jet breakup, reducing particle agglomeration, and controlling the extent of mixing and solute-solvent interaction at the point of precipitation [24][25][26].
The mechanism of precipitation in the ASES process is more complicated than for the GAS technique.In this process, mass transfer, the hydrodynamics and thermodynamics of the system influence the precipitation.It has been proposed that for precipitation under sub-critical conditions, the hydrodynamic effect is generally the dominant factor.Particles are generated by solvation of the SCF into the droplet and jet break up followed by extraction of the solvent from the droplet resulting in dry powder formation.Above the critical conditions, the organic solution is completely miscible in the antisolvent so particles are formed by nucleation and growth in the whole volume of the precipitation vessel [27].

Supercritical Fluid Drug Delivery (SFDD)
In this technique, the pulmonary drug delivery of fine aerosol particles of pharmaceutical compounds is made feasible by supercritical fluid technology [28,29].Fine aerosols of the desired substance are formed by mixing a supercritical fluid with the desired substance that is present in solution, suspension or dispersion.A gas borne dispersion of fine particles having an average diameter between 0.1 J.Lm and 6.5 J.Lm is formed after rapidly reducing the pressure of the mixture to the atmospheric pressure, which is then rapidly mixed with a large amount of air before being passed to the lung of a subject.A hand held device has been patented for the pulmonary delivery of fine aerosol particles through oral and nasal passages to humans [30,31].

Impregnation of polymers with pharmaceutical compounds
Supercritical fluids may cause swelling of polymers.Pharmaceutical compounds can thus be impregnated with a solid polymer [32,33].The supercritical fluid swells the polymer at or near supercritical conditions and causes the impregnation of the active material into the polymer matrix, examples of which include polyethylene, polypropylene, ethylene-ethyl acrylate copolymer and ethylene vinyl acetate copolymer [32].The approach can be used to develop novel controlled-release dosage forms to deposit thermolabile materials into polymers.
In a similar study of the impregnation of polymers with bioactive compounds, the loading of drugs on cross-linked polymer using supercritical fluids has been described [33].The method involved contacting the SCF containing the solubilized drug with a crosslinked polymer, resulting in impregnation of the polymer with the drug after the removal of the SCF.

Micronisation and Recrystallisation of
Pharmaceutical Compounds Fine particles of pharmaceuticals with a narrow particle size distribution are essential for the development of inhalation aerosols, injectable suspensions, controlled release dosage forms, and other specialised drug delivery systems.In addition, particle size is a critical parameter that determines the rate of dissolution of the drug in the biological fluids and hence has a significant effect on the bioavailability of the poorly water-soluble drugs for which the dissolution is the rate-limiting step in the absorption.
Micronisation and recrystallisation of pharmaceutical compounds using SCFs has many advantages over conventional techniques, such as minimum product contamination, reduced waste streams, enabling the processing of thermolabile, shock-and chemicallysensitive compounds, and the possibility of producing particles with narrow size distribution in a single step operation.
Numerous investigators have employed SCFs as alternative approaches to reduce the particle size of a broad spectrum of pharmaceutical compounds including small molecular weight compounds, peptides, proteins and polymers (Table 2, 3).
Rapid expansion of supercritical solutions has been utilised for the recrystallisation of pharmaceutical compounds.Some of the compounds that have been processed to date are listed in Table 2.In a recent study, ibuprofen, a poorly water-soluble anti-inflammatory agent, has been micronised by RESS to enhance its dissolution characteristics.A typical SEM of the RESS processed ibuprofen powder is shown in Fig. (5d) [34].The study showed that the RESS processed powder exhibited higher powder dissolution rates compared with the conventionally crystallised and micronised ibuprofen powder.Interestingly, there was no significant difference in the disc intrinsic dissolution rate between the RESS processed and the conventionally crystallized and micronised powder, thus indicating that the higher powder dissolution rate is a result of particle size reduction of the crystals and not a change in crystallinity.
Microparticles and microspheres of polylactic acid (PLA) (Mw=5,500) in the size range of 4 to 25 11m were precipitated from C0 2 and a COz-acetone mixture [5].Irregular-sized particles of PLA (Mw= 5,500) ranging from 10 to 20 11m were precipitated from C0 2 [5].The precipitation of polyglycolic acid (PGA) (Mw=6,000) from C0 2 produced needle particles of 10-40 11m length and regular particles [ 5].Gas antisolvent techniques have also been utilised for the crystallisation and micronisation of a broad spectrum of pharmaceutical compounds.Some of the compounds micronised by the various gas antisolvent techniques are listed in Table 3.
Small molecular weight pharmaceuticals have been successfully recrystallised and micronised using gas antisolvent techniques.Salmeterol xinafoate, a bronchodilator, was precipitated using the SEDS technique as micron-sized crystals with higher fine particle mass compared with the conventionally 62 crystallised and micronised product [50].Poorly water-soluble compounds have also been successfully micronised using the ASES technique [66,67] (Figure 5b, c, d).Higher powder dissolution profiles were observed for the processed powders of mefenamic acid, phenytoin, griseofulvin compared with the conventionally crystallised and micronised powders.It is concluded that although the crystallisation process in the ASES technique is instantaneous, the crystallinity of the processed powders was favourably comparable with the conventionally recrystallised and micronised powders [57,66,67].
Gas antisolvent techniques have also been used for the production of micron-sized particles of proteins suitable for inhalation delivery, such as insulin [ 18,45,47], catalase [43,46], trypsin and lysozyme [49,68].Catalase precipitated as 111m spherical and rectangular particles whereas insulin formed both agglomerated nanospheres and 1 11m thick needles 5 11m in length.Temperature, solute concentration and the nature of the solvent had little effect on particle size.The biological activity of the insulin powder was also shown by in-vivo studies to be unchanged.Raman spectroscopy further confirmed the maintenance ofthe secondary structure ofthe protein [47].
Precipitation of solutes using gas antisolvent techniques relies on the solubility of the solute in an organic solvent that is miscible with the antisolvent.The difficulty of applying gas antisolvent techniques to the processing of proteins is that they involve exposure of the protein to organic solvents, the latter being potential denaturants and very poor solvents for KONA No.l9 (2001) most therapeutic macromolecules.The ASES has been modified to enable the spraying of aqueous protein solution simultaneously with an organic solvent into carbon dioxide [24] or directly into carbon dioxide, which has been modified with an organic solvent [22,65].
Recombinant human immunoglobulin (rhiG) [65], lysozyme [22], albumin, recombinant human deoxyribonuclease (rhDNase), insulin [22], trypsin [53], a therapeutic peptide antibody Fv and Fab and plasmid DNA [54] have been precipitated from aqueous based solutions.Except for rhiG, which was not obtained as Fig. 5 SEM images of poorly water soluble compounds, mefenamic acid (a), phenytoin (b), griseofulvin (c) processed by ASES (66,67), ibuprofen (d) by RESS (34).a stable powder, all the proteins precipitated as micron-sized particles or agglomerated nano-spheres.The SEM images of the typical precipitate that is produced by the modified ASES are shown in Figure 6.Particle sizing of lysozyme by the impaction technique has shown a fine particle mass ( <5 11m) of more than 60% for the processed powder [22].Depending on the protein, the biochemical integrity of the proteins micronised using the modified ASES can be a problem.While lysozyme recovered almost complete biological activity after reconstitution in water, other proteins were denatured to various extents.
Attempts have been made to reduce the agglomeration of the precipitated particles of polymer [25].A coaxial nozzle was utilised to modify the solution injection device through which polystyrene and PLA particles were precipitated from toluene and methylene chloride (CH 2 CL 2 ), respectively [25].A stabiliser, (poly(1,1-dihydroperfluorooctyl acrylate)), was added into the solutions to further reduce particle flocculation and agglomeration [25].The addition of the stabiliser was found to significantly reduce flocculation.

Encapsulation and co-precipitation
In the development of special delivery systems like controlled release therapeutics, it is frequently desirable to disperse the material into very fine, uniform particles.Such particles may then be advantageously incorporated into controlled-release delivery vehicles such as polymeric microspheres or implantable devices, or delivered to the lung as aerosol.
Conventional methods to prepare drug-loaded microspheres include solvent evaporation, spray-drying, freeze-drying and double emulsion technique.Both drug and carrier are dissolved in an aqueous or non-aqueous solvent, which is then removed to results in polymer-coated drug particles.These methods are associated with difficulties with the complete removal of organic solvents from the end product, thermal degradation upon solvent removal by heating, low process yields, wide particle size distribution, and low drug loading.
Supercritical fluid technology is an alternative microencapsulation process, which is still in its early stages with many areas yet to be explored.The first application of the SCFs in the production of polymerdrug microspheres was described by Muller and Fischer using SC carbon dioxide as antisolvent in an ASES process [12].Microspheres of 100 micron containing clonidine hydrochloride and PLA (17000) were produced from methylene chloride solution at 60°C and 100 bar.The in-vitro release studies have shown that 50% of the drug was released after 24 hours in phosphate buffer pH 7.5 at 37°C.The potential of the ASES process for microencapsulation of pharmaceuticals has been demonstrated by several studies (Table 4).Studies have involved the simultaneous precipitation of neat core and coating solute solutions or the precipitation of pre-mixed solutions with different types of nozzles.
Various studies have shown that drug loading is a function of the solubility of the drug in the antisolvent and therefore is a function of the pressure or density of the system [72, 7 4, 78].It was generally found that the higher the solubility of the drug, the lower the drug loading due to the enhancement in solubility and thus the extraction of the drug into the antisolvent phase.Drug loadings of nonpolar drugs, such as piroxicam in PLA particles, were relatively low and decreased significantly as the pressure was increased from 90 to 200 bar at 40°C [73].Experiments with the more polar drugs, such as hyoscine-butylbromide achieved drug loadings of 19.5 wt %, which remained unchanged even at higher pressures due to the negligible change in solubility in the carbon dioxide antisolvent system [74].
Microencapsulation of ionic compounds, which are not soluble in an organic solvent suitable for ASES processing, was achieved after complexation with hydrophobic ion pairing in methylene chloride [78,79].Drug loading efficiencies of up to 37.4%, 24.7%, and 9.2% were achieved for rifampin, gentamycin and naltrexone, respectively.Similar approaches to encapsulate ionic compounds, such as a-chymotrypsin, imipramine, insulin, ribonuclease, cytochrome C, pentamidine and streptomycin have been reported [56].
The potential of the ASES process to encapsulate proteins has been investigated in few studies [76,77,82].In an attempt to encapsulate lysozyme with L-PLA, or PLGA from a dichloromethane solution [82], a suspension of lysozyme in the polymer solution was injected into the carbon dioxide antisolvent existing as a vapour to partially solidify the droplets before they fell into a carbon dioxide liquid phase.The integrity of the polymer coating or the biological activity of the lysozyme was not investigated.
The effect of polymer crystallinity and thermal behaviour on the encapsulation of two model drugs, albumin and estriol, have been investigated [77].The drug content and the release studies confirmed that the blocked copolymers b-poly-L-lactide-co-D,Llactide-co-glycolide and poly-D,lrlactide-co-glycolide in the microparticles gave the same protein content, which was mainly on the surface or immediately below.
The feasibility of utilising RESS for encapsulation of
drugs in DL-PLA (Mw=lO,OOO) has been demonstrated.Microparticles consisting of lovastatin crystals coated with DL-PLA polymer were formed by combining the extraction of drug and polymer into one stream prior to expansion [8].Precipitates with high lovastatin concentration (>30%) showed a network morphology while those with low concentration showed microparticle and microsphere morphology.
The particles consisted of drug needles coated with polymer.
In a similar study, the suitability of RESS as an encapsulation procedure was demonstrated by the coprecipitation of naproxen and L-PLA (Mw=2,000) [9].The drug and polymer were pre-mixed and loaded in the same extraction vessel, a capillary nozzle was used as an expansion device.Composite particles containing microcrystalline naproxen particles at the core, with coated polymer on the surface, were observed.
Supercritical carbon dioxide was also employed in the inclusion of drug compounds in cyclodextrin molecules using the RESS technique.A piroxicam-13-cyclodextrin inclusion complex was prepared to enhance the dissolution characteristics of poorly water-soluble piroxicam [ 83].

Co-precipitation and Mixing of powders
Mixing of powders can be achieved by co-precipitation of the solutes of interest using SCF.The application of RESS was extended to the processing of a homogeneous mixture of powders in a single step process.Mixing of active ingredients and excipients was demonstrated using the RESS process [84].The method involved solubilising and mixing the active ingredients and the excipients in the liquid carbon dioxide under a pressure sufficient to maintain carbon dioxide in liquid state (20°C and 700-900 psi).Depressurisation of the solution resulted in the coprecipitation of both the active ingredients and the excipients.
The co-precipitation of caffeine and 13-carotene using two separated extraction vessels in a RESS process has been reported [85].Under the same conditions, 13-carotene precipitated as shorter needles compared to caffeine.The results also showed the ultimate mixing of both caffeine and carotene needles with no aggregation, and the precipitates were separated from each other.More caffeine precipitates were observed in the mixture due to the higher solubility of caffeine in C0 2 compared to that of 13carotene.The ratio of caffeine to 13-carotene precipitates was approximately 16.It was concluded that the 66 homogeneity of the mixture precipitated by RESS in this study could not be obtained by mechanical mixing.
The ASES process was also utilised in the production of powder mixtures of pharmaceutical compounds.Combinations of urea/ chloramphenicol and ascorbic acid/paracetamol have been co-precipitated from ethanol at 40°C and 90 bar using the ASES process [86].The quality of the products produced each time was not quantified, although problems with particle agglomeration and fractionation below 85 bar were identified for the co-precipitation of paracetamol with ascorbic acid.

Liposome Formation
Liposomes are microscopic vesicles in which an aqueous volume is entirely enclosed by a membrane normally composed of single or multiple phospholipid bilayers.Liposomes can entrap hydrophilic or lipophilic compounds so they are used as carriers or vesicles for the delivery of therapeutic compounds.Liposomes can be made with different features, which can enhance drug efficacy, reduce toxicity, and prolong the therapeutic effect.Because of their similarity to natural cells, liposomes have been reported to be ideal carriers for parenteral administration.
Conventional techniques of liposome formation include methods that involve the use of organic solvents or detergents, which often leave residues in the end product, or methods which involve the use of high pressure extrusion of the liposome through polycarbonate filters of controlled pore size.Furthermore, liposome sterility is accomplished by independently sterilizing the component parts by autoclave or filtration and then mixing in a sterile environment.This process is difficult, time consuming, and expensive.In addition, sterilization techniques may cause damage or alter the features of the multilayered liposomes.
Formation of liposomes containing drugs using supercritical technology is a new application that has been reported recently [87,88].Supercritical fluid methods and apparatus capable of forming liposomes that carry hydrophobic drugs, have been described [87].One method of forming liposomes containing the drug is referred to as the decompression method.The method involves forming a solution or mixture of a phospholipid, a hydrophobic drug, an aqueous medium, and the supercritical or sub-critical carbon dioxide, which is then decompressed to form the liposomes.The other method is referred to as the injection method of forming liposomes.In contrast to the decompression method, the injection method does not involve pressurisation of the aqueous phase.Instead, the solution or the mixture of the drug, a phospholipid and the supercritical or sub-critical C0 2 , is injected through a tip or orifice into an aqueous phase.At the time of injection or afterwards, the solution or the mixture is decompressed to form the liposomes containing the drug.
Preparation of liposomes encapsulating water-soluble compounds using supercritical carbon dioxide has also been reported [88].The encapsulation method involves dissolving the lipid components (1-palmityl-2oleoyl phosphatidylcholine (PoPe) and cholesterol, 7:3 molar ratio) under pressure in supercritical carbon dioxide.The homogenous supercritical solution is expanded and simultaneously mixed with the aqueous phase containing the water-soluble compound to form liposomes encapsulating the compound.Liposomes with average diameter of 200 nm with an encapsulating efficiency of 20% could be reproducibly prepared.The amount of organic solvent used compared to conventional preparation methods could be considerably reduced.

Scale up
The experimental scale of the majority of the gas antisolvent studies has involved high-pressure precipitation vessels of about 50-100 mL in volume, with a sight gauge to enable visual observations during operation.The ASES process has been scaled up to a 50-L precipitation vessel that has been used in the generation of microparticles of PLA [71].Recently the SEDS particle formation process has been scaled from a 50 mL laboratory vessel through a pilot plant of 1-10 Land to a small manufacturing plant.It was reported that the size and solid state properties of each scale of manufacture are comparable to the targeted mean sizes ranging from sub-micron to 20 1-lm [ 89].A production plant for gas antisolvent crystallisation was scaled up to a 250 L crystalliser in which the drug solution is fed into the crystalliser via a nozzle system with a consumption of 2-3 kg antisolvent per kg of solid product [86].

Conclusions
Supercritical fluids provide a promising option for designing stable protein formulations.They also offer alternative means of processing some peptides and proteins without affecting the activity and integrity of the molecule.Supercritical fluid technology provides a processing option for size reduction and recrystallisation of pharmaceuticals in a single step operation.It also provides a solvent free alternative to traditional microencapsulation techniques.The research on protein encapsulation is still in its infancy.For coating operations, the solid content in coating solutions can be significantly increased by employing supercritical fluids.
Rapid expansion of supercritical fluids and supercritical antisolvent recrystallisation are techniques which are capable of generating micron-sized particles suitable for special delivery systems.Many factors have to be considered in the selection of the appropriate technique.Based on the need for reasonably high drug solubilities in SCFs to make RESS commercially viable, it appears that the antisolvent recrystallisation techniques may be more broadly applicable.
Despite recent advances in the development of SCF techniques, fundamental studies on phase behaviour and mass transfer information that are required to obtain a reliable picture of the process are lacking.Models validated on reliable sets of experimental data are also required to enable the scale-up to industrial scales.However, the process has already proved to be very effective in several fields.The achievements so far will be the driving force for further effort in scaling up this technology.

Neil R. Foster
Neil Foster received his B.S. and PhD in Chemical Technology from the University of New South Wales.His PhD research involved heterogeneous oxidation of aromatic organics.Following his graduation he completed a postdoctoral year at McMaster University, Canada, where he was involved in polymer reaction engineering.In 1979 he returned to Australia to take up an appointment with CSIRO where he was involved in the Fossil Fuels Liquefaction program which involved high temperature, high pressure reaction engineering.Neil returned to the University of New South Wales in 1986 and is currently Professor of Chemical Engineering and leads the UNSW Supercritical Fluids Research Group.The major focus of current work is the formulation of novel delivery systems for therapeutic drugs using dense gas technology.Neil has published more than 150 scientific and technical papers and is currently working closely with an industry partner, Eiffel Technologies Limited (a publicly listed Australian company) to develop and commercialise dense gas technology applications for processing of pharmaceuticals.

4 A
Fig.4A schematic diagram of the aerosol solvent extraction system apparatus.
: precipitation with compressed antisolvents : poly(glycolic acid) : poly(lactic acid) : poly(lactide-co-glycolic acid) : rapid expansion of supercritical solution : recombinant human deoxyribonuclease : recombinant human immunoglobulin : supercritical antisolvent : supercritical fluid : solution-enhanced dispersion by supercritical fluids : supercritical fluid drug delivery : supercritical fluid technology : boiling point temperature : critical temperature : tetrahydrofuran.I Author's short biography I Rana T. Bustami Rana Bustami received her B.S. in pharmacy from University of Jordan.Following her graduation, she joined the Arab Pharmaceutical Manufacturing Co. where she was involved in the development of stability indicating assays and then in the development of sustained release dosage forms for wide range of pharmaceuticals.She received her graduate diploma in science from the University of Sydney in 1997.Rana has recently submitted her PhD Thesis titled The Application of Supercritical Fluid Technology in the Micronisation of Pharmaceuticals.Hak-Kim Chan For photo & biography, please refer to the accompanying article to KONA "Pharmaceutical Dry Powder Aerosol Delivery" by Nora YK Chew & Hak-Kim Chan to be published in this volume (No.19, 2001) Fariba Dehghani Fariba Dehghani received her B.S. and M.S. in chemical engineering from the Shiraz University in Iran.For her Masters she worked on the "Evaluation of CORGC Equation of State for Prediction of Thermodynamic Properties of Polar and Nonpolar Compounds" under the direction of Prof. M. Moshfeghian.She received her PhD in chemical engineering from the University of New South Wales in 1996 under the supervision of Prof. Neil R Foster concerned the "Extraction of Rare Earth Metals Using the Dense Gas Process".Following her graduation she continues research on application of dense gases for processing fine chemicals as a senior research scientist at the University of New South Wales.Her research involves sterilization, micronisation I crystallization of pharmaceutical compounds, study the solute-solvent interaction in supercritical fluids.

Table 1
[1].Carbon dioxide is a commonly used supercritical fluid in pharmaceutical applications as it is nonflammable, inexpensive, has a moderate critical temperature (31.1 °C) and is relatively non-toxic.

Table 1
Critical parameters for solvents that can be used as supercritical fluid [1].

Table 2
Particle size reduction of pharmaceuticals using rapid expansion of supercritical fluids.

Table 3
Particle size reduction of pharmaceuticals using the aerosol solvent extraction system technique.

Table 4
Encapsulation of pharmaceutical compounds using the aerosol solvent extraction system technique.