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178 Recent Patents on Drug Delivery & Formulation 2009, 3, 178-192 Recent Patents Review in Microencapsulation of Pharmaceuticals Using the Emulsion Solvent Removal Methods Wasfy M. Obeidat* University of Sharjah, University of Sharjah - College of Pharmacy, P.O. Box: 27272, Sharjah - United Arab Emirates Permanant Address: Jordan University of Science and Technology, Irbid 22110, P.O. Box 3030, Jordan Received: March 16, 2009; Accepted: June 5, 2009; Revised: June 29, 2009 Abstract: Several methods and techniques are potentially useful for the preparation of polymeric microparticles in the broad field of microencapsulation. The preparation method determines the type and the size of microparticle and influence the ability of the interaction among the components used in microparticle formulations. This review is devoted to describe and allocate the recently awarded and pending patents regarding the technical and formulation innovations in microparticles involved in drug delivery that are based mainly on the emulsion solvent removal methods. The term microparticle designates systems larger than one micrometer in diameter and is used usually to describe both microcapsules and microspheres. Microparticles-containing drugs are employed for various purposes including -but not restricted to- controlled drug delivery, masking the taste and odor of drugs, protection of the drugs from degradation, and protection of the body from the toxic effects of the drugs. Polymeric carriers being essentially multidisciplinary are commonly utilized in microparticle fabrication and they can be of an erodible or a non-erodible type. Keywords: Microspheres, microcapsules, emulsion, evaporation, extraction, hydrophilic, hydrophobic, drugs, surfactants, polymers, degradable, lipids, monosize, proteins, starch. 1. INTRODUCTION Microencapsulation is one of the most interesting fields in the area of pharmaceutical technology since its inception many years ago. It is an interdisciplinary field that requires knowledge of polymer science and familiarity with emulsion technology. Starting first as an art than a science, nowadays the topic of microencapsulation is extensively studied inside major pharmaceutical companies and universities as well as research institutes. Polymeric drug delivery devices are focusing on the encapsulation of large molecules, e.g., peptides, proteins, and DNA/RNA for potential use as vaccines or as long-acting release drug formulations. Importantly, some of these initiatives led to important pharmaceutical products and most of them are still on the market (e.g., Lupron Depot®, Zoladex®, Decapeptyl®, Eligard®, Enantone®, Trenantone®, Nutropin Depot®, and Profact®) [1]. In addition, encapsulation for controlling the release of highly water soluble drugs received much attention. This article reviews the current state of the art in emulsion solvent evaporation/extraction based microencapsulation technologies. It is focused on the formulation, optimization and the drug delivery related aspects rather than equipments and machinery, as described in the most recent and early pioneered patents and the most related articles in the literature. Both, well-established and more advanced technologies are reviewed. Microencapsulation processes based on solvent removal from emulsions have been reviewed in various books [2-4] and review articles [5-10]. 2. DEFINITION AND RATIONAL FOR MICROENCAPSULATION Microencapsulation products (microparticles) can be defined as small entities that contain an active agent or core material surrounded by a shell or embedded into a matrix structure. Most Microparticle shells or matrices are organic polymers, but lipids and waxes are also used. It is generally accepted that microencapsulation products (microparticles) are larger than 1 micrometer in diameter and can be up to 1000 micrometers. Commercial microparticles have a diameter 3 and 800 micrometers and contain 10-90% w/w core. A wide range of core materials has been encapsulated, including adhesives, agrochemicals, live cells, active enzymes, flavors, fragrances, pharmaceuticals and ink. Morphologically, two general structures exist: microcapsules and microspheres [4]. A microcapsule is a reservoir-type system with regular or irregular shapes that contains a welldefined core and envelope. The core can be solid, liquid, or gas; the envelope is made of a continuous, porous or nonporous, polymeric phase created by one or more polymers. Alternatively, a microsphere is a homogeneous or monolithic structure made of a continuous phase of one or more miscible polymers in which particulate drug is dispersed throughout the matrix, at either the macroscopic (particulates) or molecular (dissolution) level. However, the difference between the two systems-microcapsules and microspheres- is the nature of the microsphere matrix, in which no well-defined wall or envelope exists [2]. Different methods of encapsulation result, in most cases, in either a microcapsule or a microsphere. For example, interfacial polymerization and coacervation methods almost always produce a microcapsule, whereas solvent evaporation may result in a microsphere or a microcapsule, depending on the formulation and processing factors. However, novel methods © 2009 Bentham Science Publishers Ltd. *Address correspondence to this author at the University of Sharjah, University of Sharjah-college of Pharmacy, P.O. Box: 27272 SharjahUnited Arab Emirates; Tel: +971 6 505 7414; E-mail: [email protected] 1872-2113/09 $100.00+.00 Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 179 have been designed to use solvent evaporation to create in one step a double-walled microsphere, which is essentially a microcapsule [11]. The technique of microencapsulation by emulsion solvent removal method has been applied extensively in pharmaceutical industries for various purposes such as controlled drug delivery, masking the taste and odor of drugs, protection of the drugs from degradation, and protection of the body from the toxic effects of the drugs. Irrespective of the preferred site of drug delivery, controlled drug release systems are known to have many advantages over conventional ones [6, 7]. It is well known that patient compliance is better when the drug dosing is only once or twice daily. It has been reported that, as the number of doses per day increases, there is a greater risk that the patient will either forget or neglect to take every dose [12]. Other major advantages are the optimization of drug concentration in plasma and reduction of side effects, particularly for drugs with low therapeutic indexes. For oral administration, advantages of multiple-unit products include ready distribution over a large area, less variable release and release which is less dependent on gastric transit time [13]. This potentially improves drug absorption and reduces local irritation to the GI mucosa [14]. 3. PRODUCTION OF MICROPARTICLES USING THE EMULSION SOLVENT REMOVAL METHODS 3.1. Advantages of the Emulsion Solvent Removal Method There are increasing numbers of encapsulation processes. Some are based exclusively on physical pheno-mena. Others combine physical and chemical phenomena. Many new patents evolve solely on the basis of novel ways to produce microspheres. Interfacial polymerization, simple and complex coacervation, polymer-polymer incompatibility and phase separation, hot melt, spray drying and salting-out are among these processes [2, 9]. However, the emulsion solvent removal is the oldest and most widely used method to accomplish encapsulation. Unlike spray drying method where the temperature-sensitive compounds are degraded and control of the particle size is difficult [15], the emulsion solvent removal method does not require elevated temperatures. Contrary to phase separation and coacervation methods which are impaired by residual solvents and coacervating agents found in the microspheres [16], the emulsion solvent removal method is deprived of phase separation inducing agents. Emulsion solvent removal method allows for the creation of microparticles or nanoparticles that have a more optimized release of the encapsulated material. For example, if the insoluble particle is localized to the surface of the microparticle, the system will have a large burst effect. In contrast, creating a homogeneous dispersion of the insoluble particle within the polymeric matrix will help to create a system with release kinetics that begin to approach the classical zero-ordered release kinetics which are often perceived as being ideal in the field of drug delivery [4]. 3.2. Emulsion Formation and Fabrication Materials Used in solvent removal method (and in all emulsion based methods) as well as most of the other encapsulation methods involves the formation of an emulsion usually of a polymeric solution inside a continuous phase where the drug is dispersed or solubilized inside this polymeric solution. The particle size in the final encapsulation product is primarily determined by emulsion formation [9]. An emulsion is achieved by applying mechanical energy which deforms the interface between the two phases to such an extent that droplets form. These droplets are typically large and are subsequently disrupted or broken down into smaller ones. The droplet formation step determines the size and size distribution of the resulting microspheres. Microsphere size may affect the rate of drug release, drug encapsulation efficiency, product syringeability, in vivo fate in terms of uptake by phagocytic cells and biodistribution of the particles after subcutaneous injection of intranasal administration [4]. The ability to disrupt the larger droplets is a critical step in emulsification and in encapsulation where an emulsion is prepared. A suitable surfactant acceptable for therapeutic use is needed to produce a stable emulsion. Many devices have been designed to produce emulsions depending on the desired particle size. For large scale production of emulsions, colloidal mills, and high pressure homogenizers are most often used [9]. Stirring is the simplest and most straightforward method that is exclusively used in laboratories to generate droplets of the emulsion. Increasing the mixing or stirring speed generally results in decreased microsphere mean size [17]. In addition, more vigorous mixing also results in lower microsphere polydispersity. The extent of size reduction that is attained depends on the viscosity of the disperse and continuous phases, the interfacial tension between the two phases, their volume ratio, the geometry and number of the impeller(s) and the size ratio of impeller and mixing vessel [18]. The surface active agents or viscosity-enhancing stabilizers such as PVA and polysorbates that are generally added to the continuous phase prevent coalescence of the emulsion droplets. Increasing the stabilizer concentration frequently leads to decreased microsphere sizes. With macromolecular stabilizers, the viscosity of the continuous phase will also increase, amplifying for a given stirring rate the shear forces acting upon the emulsion droplets and thus minimizing their sizes [18, 19]. The effect viscosity of the polymer solution phase on the resulting microsphere sizes has been investigated [20, 21]. Results indicated that an increase in the agitation intensity was required to decrease the produced microsphere diameters when polymer solution phase of higher viscosity was used. The continuous phase volume ratio seems to affect the particle size of the produced microspheres. Various studies reported a reduction in the mean microsphere size with decreasing continuous phase volume [19, 22, 23]. Owing to their excellent biocompatibility, the biodegradable polyesters poly (lactic acid) (PLA) and poly(lactic-coglycolic acid) (PLGA) are the most frequently used biomaterials for the microencapsulation of therapeutics and antigens [24, 25]. Other materials like proteins [26], polysaccharides such as chitosan [27-29], and lipids [30-32] have also been studied, although at a lower frequency. An understanding of the properties of emulsions is extremely important since the first step in the emulsion 180 Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 Wasfy M. Obeidat 3.3. Microencapsulation Process and Drug Incorporation There are several techniques that use microencapsulation by the emulsion solvent removal methods. The choice of certain technique that will give rise to an efficient drug encapsulation depends on the hydrophilicity or the hydrophobicity of drug. Generally, in the emulsion solvent removal method, polymers and oligomers are dissolved in a suitable volatile organic solvent to form polymeric solution phase that is immiscible with the continuous aqueous phase containing a surface active agent as a stabilizer. The capacity of the continuous phase is insufficient to dissolve the entire volume of the disperse phase solvent. The medicament is usually dispersed as finely pulverised solid or dissolved in the polymeric solution phase and the resulting solution or dispersion is then emulsified with the continuous phase under agitation to form discrete droplets of the (o/w) type suitable for the encapsulation of insoluble or poorly watersoluble drugs [33, 34]. Entrapment of small molecules and solid protein particles was shown to be improved with decreasing particle size [35, 36]. Increasing the volume fraction of the internal aqueous phase lowered the encapsulation efficiency due to droplet coalescence and increased the probability of contact between the internal drug solution and the external extraction phase resulting in drug loss [37]; in addition, an increase in the burst release and microsphere porosity was reported [38]. The size of the oil phase droplets, that determines the size of the microspheres produced, is dependent on the speed the system is agitated when the oil phase is added to the aqueous phase and the stabilizer type and concentration. Generally, a continuous phase that is a non-solvent for the microencapsulated bioactive compound is favourable. While for lipophilic compounds, aqueous solutions may be comfortably chosen, the use of hydrophobic, organic liquids as continuous phase for the encapsulation of hydrophilic compounds is more delicate [39-41]. Once the emulsion is stabilized, agitation is maintained and the solvent present in the internal phase must first diffuse into the continuous phase and then almost completely evaporates/extracted at the water/air interface aiding the hard microspheres formation. Thus, a precipitation of small polymer particles from an o/w emulsion occurs. The rate of volatile solvent removal from the solidifying microspheres can be controlled by the temperature of the microsphere dispersion. Figure (1) shows a scanning electron micrograph of matrix microspheres prepared using the emulsion solvent evaporation method, while Fig. (2) shows a cross sectional view for cleaved microspheres prepared using the same method. Higher temperatures will facilitate the evaporation of the solvent from the continuous phase and thereby maintain a high concentration gradient for the solvent between the microspheres and the continuous phase [38, 42]. As an alternative to elevated temperatures, reduced pressure is sometimes used to promote the evaporation of the solvent [43]. The solvent evaporation method was described very early by Vrancken and Claeys [44], Kitajima et al. [45], Schnoring et al. [46], Fukushima et al. [47], Morishita et al. [48], Tice and Lewis [49]. In these patents a material to be encapsulated is emulsified in a solution of a polymeric Fig. (1). Scanning electron micrograph of cellulose esters (CAB381-20) matrix microspheres 250-355 mm sieve fraction prepared using the emulsion solvent evaporation method with. (From: W. M. Obeidat, J. C. Price, J. Microencapsulation 2004; 21: 47-57). Fig. (2). Cross-sectional view of a cleaved microsphere preparation from a 300-425- m sieve fraction after buffer dissolution (From: W. M. Obeidat et al. AAPS PharmSciTech. DOI: 10.1208/s12249009-9240-3. material in a solvent which is immiscible with water and then is emulsified in an aqueous solution containing a hydrophilic colloid to form an o/w emulsion. The emulsion solvent removal process is conceptually simple, the physicochemical phenomena governing this process are complex due the existence of a large number of processing and formulation variables which can profoundly affect the nature of the product obtained [6]. Some of the important formulation factors affecting the final particle’s characteristics include the solvent; which should be able to dissolve the chosen polymer, but poorly soluble in the continuous Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 181 phase and it should be of high volatility, low boiling point and low toxicity. Other factors can include,-the surfactants such as non-ionic, anionic and cationic agents, the concentration of the used polymer or polymer combination and the amount of the aqueous phase [7, 10]. Parameters and operating conditions include the viscosity of the dispersed phase, quantity of active material (drug) in the dispersed phase, agitation intensity, the geometry of the agitator, the number of impellers and their position and the ratio of impeller’s diameter compared to the agitator’s diameter geometries, pressure and temperature. In addition, one of the requirements of the emulsion solvent removal process is that the active agent (i.e., drug) partitions favorably into the oil phase [4]. However, this method is generally suitable for the encapsulation of hydrophobic compounds and is not suitable for high hydrophilic drugs since the drug may not be dissolved in the organic solvent and/or may diffuse into the continuous phase during emulsification, leading to a great loss of drug. Various hydrophobic compounds have been encapsulated using this method. Beck, Tice, and coworkers were among the first to intensively study the encapsulation steroids and to focus on their efficiency in vivo [50-54]. Later on, large numbers of publications and patents have been published. Hughes and Olejnik [55] provided a method of sustained delivery of an active drug to a posterior part of an eye of a mammal to treat or prevent a disease or condition affecting mammals. The method is comprised of administering an effective amount of an ester prodrug of the active drug such as tazarotene (prodrug of tazarotenic acid) subconjunctivally or periocularly since a systemic administration requires high systemic concentration of the prodrug. The ester prodrug is contained in biodegradable polymeric microparticle system prepared using the o/w emulsion solvent evaporation methods. Lee et al. [56, 57] prepared a composition in the form of thin film or strip composed of microspheres containing antibiotic such as minocycline HCl and made with biodegradable polymer prepared by a modified o/w emulsification technique followed by solvent evaporation. Water-soluble polysaccharide polymers such as pectin was used for making thin film or strip containing microspheres intended for local sustained release administration into the periodontal pocket. The thin film or strip is coated by spray-coating with cation salt aqueous solution of calcium or barium chlorides. A method for producing sustained release microsphere preparation for water-soluble medicament, which has high incorporation efficiency of the medicament and low initial burst was described by Kobayashi et al. [58]. First; the formation of a solid dispersion of the water-soluble active ingredient such as TRH derivative into the biodegradable polymer at molecular level by dissolving these substances in one or two solvents in which both can dissolve followed by removing the solvent. Secondly, the produced solid dispersion is dissolved an organic solvent being water-immiscible and adding the resulting oil phase into an aqueous phase containing emulsifying agent to give o/w emulsion, and followed by removing the organic solvent from the oil phase of the resulting emulsion. In one embodiment, Traynor et al. used the o/w emulsion to produce sol-gel microcapsules (containing sunscreens) that are highly positively charged using non-ionizing cationic additives which can include cationic polymers [59]. An injectable slow-release partial opioid agonist or opioid antagonist in a poly (D, L-lactide) microspheres with a small amount of residual ethyl acetate was provided by Tice et al. [60] and Markland et al. [61] where an o/w emulsion is first prepared from an organic phase made of ethyl acetate and an aqueous phase comprised an aqueous ethyl acetate containing solution of poly(vinyl alcohol). Microspheres are recovered by extraction with water. Wen and Anderson [62] prepared single wall biodegradable microspheres by extracting an o/w emulsion containing steroidal and non-steroidal anti-inflammatory agents. Otherwise, double wall microspheres were prepared. Microspheres containing the active were then immobilized on a substrate surface in a polymeric matrix that is an implantable medical article or an in situ formed matrix. Solidification method of the hydrophilic capsule materials such as gelatin can be through rapidly lowering the temperature and subsequent dehydration. While such method achieved some significant commercial success, difficulties have sometimes been encountered in rapidly inducing solidification of the microencapsulating material. However, Lee and Yuk [63, 64] used emulsification, chelation, and freeze-drying to prepare microcapsules of oil droplet containing drug for oral administration using biodegradable and biocompatible natural polymer such as polysaccharide such as sodium alginate which has a metal chelating capacity. The drug dispersed in oil phase is added to the aqueous solution mixture (to be used as capsule material) of polysaccharide which has metal chelating capacity, biocompatible and water soluble polymer and emulsifying agents to produce an o/w emulsion. As soon as possible after formation of the emulsion, it is added to multivalent cationcontaining solution to harden the capsule material. As an alternative to elevated temperatures, reduced pressure was used by Masao et al. [43] and by Chung et al. [65] to promote the evaporation of the solvent [65]. Gardner [66] described the preparation of micro-capsules using nonemulsion solvent evaporation. In the process, polylactide (PLA) microcapsules are produced after dissolving PLA in a mixture of two miscible organic liquids, the higher vapor pressure liquid with solvent power for PLA and the second having little or no solvent power for PLA. The solution is prepared such that it is near its saturation point for PLA. Encapsulation of dispersed core material is achieved by vaporizing the liquid having solvent power for PLA causing phase separation of the PLA, then transferring the separated dispersion to an organic liquid having no solvent power for the PLA in order to harden the encap-sulating coating. The disclosed process enables forming true microcapsules or reservoir-type devices encapsulating liquid core material particularly aqueous liquid core material and o/w emulsion or liquid suspensions. Fong [67] used carboxylic acid salt surfactant to stabilize the o/w emulsion such as straightchain fatty acids, coconut fatty acids, N-acylated sarosine, or N-acylated bile acids, e.g., glycocholic acid and taurocholic acid and their salts produce microspheres using the emulsion solvent evaporation method. The essential feature of this invention is the vacuum distillation that is used to slowly remove the polymer solvent from the microdroplets in the emulsion. Strategies permitting a reduction in the loss of 182 Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 Wasfy M. Obeidat water-soluble drugs have been proposed to increase microencapsulation yields. Loss of the drug compound can be prevented by increasing the concentration of the matrix material solution; the resulting higher viscosity restricts the migration of the drug from the solidifying microspheres to the external phase by means of lowered diffusion [68-74]. In a modification to improve the percentage of incorporation of a drug in microspheres produced by the o/w solvent removal method, Yamakawa et al. [75] prepared microspheres containing neurotensin peptide at high concentration by o/w solvent evaporation method using an internal phase consisting of mixed solvent of at least one water insoluble solvent and at least one water miscible solvent with at least one glycerin fatty acid ester or propylene glycol fatty acid ester and an aqueous outer phase containing polyvinyl alcohol (PVA). Microspheres are prepared due to the quick phase separation within the oil droplets after the water miscible solvent being partitioned into the outer aqueous phase. Other means of preventing loss of the drug material into the continuous phase encompass the adjusting of the continuous phase pH to lower the solubility of the drug compound [76], or the addition of electrolytes to increase the osmotic pressure of the continuous phase [77, 78]. In addition, the rate of solvent removal can be optimized to reduce drug loss. For example, fast microsphere solidification will be preferred if the drug easily partitions into the continuous phase. On the other hand, slow solidification favors denser over more porous microspheres, affecting the drug release. The water solubility of the drug can be reduced by chemically modifying it to a lipophilic prodrug prior to its incorporation in the organic phase to allow for higher microencapsulation yield [79]. Alternatively, modification of the continuous phase of the emulsion to reduce leakage of the drug from the oily droplets can be employed. Several investigators have therefore suggested saturating the continuous phase with the drug [80, 81], replacing the continuous phase with a less hydrophilic one as in the case of the non-aqueous emulsion of oil-in-oil (o/o) type [82]. Mosier [83] developed methods for making microspheres to deliver water-soluble diagnostic and therapeutic substances such as anticancer agents. The method involves dissolving the drug and the matrix material in a volatile organic solvent with a relatively high dielectric constant (above 15), in which they are mutually soluble, then dispersing this mixture in a second volatile organic non-polar solvent with low dielectric constant (below 5) that it is immiscible with the first solvent and comprising the continuous phase leading to formation of a stable o/o emulsion with the aid using suitable surface active agents. Filtration, decanting, filtration or centrifugation can be used, to remove the major portion of the second solvent, the remaining portion of the second solvent and the first solvent can be removed by evaporation under reduced pressure the resulting microspheres are recovered in solid form. Obeidat and Price [84] employed a one step method for the preparation of microspheres having enteric and controlled release characteristics in one embodiment and swelling and controlled properties in an other using the nonaqueous solvent evaporation method comprising (o/o emulsion) followed by solvent evaporation. Microspheres were especially useful for delivery of moderately non-polar active ingredients but can be formulated to deliver very soluble polar compounds. Delgado [85] developed a method for preparing enteric polymeric microparticles containing a proteinaceous antigen in a single or double emulsification process in which the enteric polymer acts as a stabilizer for the microparticles which are formed in the process. Illum et al. [86, 87] prepared an adhesive delivery system consisting of cross linked albumin or PLGA microspheres prepared using w/o emulsification method followed by fibrial bacterial adhesion material obtained from E. coli is attached to the microsphere surfaces by adsorption or by covalent linkage. Muranishi et al. [88] produced polylactic acid microspheres having an average particle diameter of about 0.1 to 10 m and loaded with an anti-cancer agent. The process involves the steps of dissolving the drug substance and polylactic acid in a solvent, emulsifying the solution in a non-solvent, while treating the solution with ultrasonic wave, thereby forming an o/w or o/o emulsion of fine particles, and distilling away the first solvent. Prior to the application of ultrasonic wave, the solution and non-solvent may be mixed using a propeller stirrer or turbine stirrer. Garces and Viladot [89, 90] prepared matrix microcapsules by forming an aqueous matrix containing the active ingredient by heating an aqueous solution comprised of a gel former selected from hetero-polysaccharides such as agaroses, agar, pectins, gelatin and xanthans or proteins such as gelatin. The aqueous matrix is then dispersed in an oil phase to form w/o emulsion before contacting the dispersed matrix with an aqueous solution of an anionic polymer selected from the group consisting of a salt of alginic acid and an anionic chitosan derivative and removing the oil phase. The use of various gel forming proteins (collagen and gelatin) and polysaccharides (agar, calcium alginate, and carrageenan) introduced a milder, biocompatable immobilization or isolation system. Lencki et al. [91] used a w/o emulsion type for the production of microspheres obtained by mixing a hydrophobic liquid, such as a vegetable oil into water slurry containing an immobilizing agent of polyanionic polymer such as polysaccharides and a gelling agent to the immobilizing agent, leading to the formation of a dispersion of droplets of the water slurry in the hydrophobic liquid. In other words, the droplets containing oil droplets were entrapped in the immobilizing agent. The droplets gel to form microspheres by adding an oil soluble organic acid such as acetic acid. The preparation of microspheres from w/o emulsion or from o/o emulsion using the solvent evaporation method works best to incorporate a biologically active substance into microspheres. However, it is difficult to completely remove the large volume of solvents from microspheres, and there are other problems related to the safety of the operation and environmental problems. Besides, there is used a mineral oil or vegetable oil as an external oil phase in w/o emulsion and o/o emulsion, and hence, it is difficult to collect or to wash the resulting microspheres, and the remaining oil in microspheres is a significant problem. Yet further innovative methods have been proposed for the efficient encapsulation of water soluble drugs by the Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 183 emulsion solvent evaporation technique involving the formation a double emulsion (multiple emulsions) where an aqueous core material solution is emulsified in a polymervolatile organic solvent solution. The resulting emulsion, which is called the primary emulsion, is emulsified in water giving a double emulsion of w/o/w type. The organic phase acts as a barrier between the two aqueous compartments preventing the diffusion of the drugs towards the external aqueous phase. Evaporation of the volatile solvent yields a solid microcapsule with an aqueous core. Since the external phase is an aqueous solution, there is no problem as mentioned in w/o method or o/o method. However, the pharmaceutical active ingredient in oil phase often dissolves out into the external aqueous solution so that the incorporation efficiency of the active ingredient into microspheres becomes low. In addition, this process is particularly suitable for the encapsulation of drugs in low doses, which are strongly water soluble but still requires complex steps. LHRH analogue, leuprolide acetate was prepared and marketed using this method [92]. Conventional double emulsion solvent extraction or solvent evaporation methods are limited to solvents that are not too hydrophilic so that the emulsion can be formed and are not too hydrophobic so that emulsion droplets may not stay too long in a liquid state. Thus, solvents that could be successfully used in conventional methods were limited to only a few solvents; practically to methylene chloride. This is one of worst disadvantages of conventional emulsion methods, since methylene chloride is a possible carcinogen and its residual amount should be strictly controlled under the limit. The presence of residual stabilizers on the resultant microparticles is also a potential problem. The double emulsion requires many steps, rigid control of temperature and viscosity of the inner w/o emulsion, and it is difficult to encapsulate high concentrations of hydrophilic drugs. If the experimental parameters are not adequately adjusted, a wide size distribution will be obtained. Hyon and Ikada [93] used an o/o, w/o or w/o/w emulsion types followed by solvent evaporation for encapsulation of water soluble physiologically active substances in biodegradable polymers such as polylactic acid enabling a stable sustained release of the active substance over a long period of time. Microspheres with encapsulated or covalently bonded biologically active agent allowed provision of an injectable suspension as a substitute for surgical implantation and facilitate administration of multiple drugs in a single injection. These microspheres provided an initial burst to reach a therapeutic concentration followed by a zero-order release of drug to maintain the therapeutic level by compensating for metabolic loss. Wu et al. [94] prepared a biodegradable microsphere with surface hydrophilicity (without further surface modification with a hydrophilic polymer). Microspheres are formed by w/o/w emulsification-solvent evaporation technique from a double-bond-functionalized glycerol poly( caprolactone) maleic acid (PGCLM) which provides enormous potential for altering the surface chemistry of the microspheres to tailor to specific clinical applications. Ogawa et al. [95] provided a method to encapsulate watersoluble agents from w/o/w emulsions using high viscosity polymer phase and a drug retaining substance in the inner water phase to retain the drug during evaporation of the polymer solvent or by thickening the inner phase of a w/o emulsion making a w/o/w and subjecting the emulsion to an “in water drying” process [71, 96]. Drawbacks of this process include the necessity to use a thickening agent in a multi-step procedure; two emulsification steps and a drying step. An increase in the rate of solvent removal from microspheres in “in-water drying” and a reduction of the amount of solvent in microspheres containing leuprorelin acetate or thyrotropin-releasing hormone using a w/o/w emulsification process were provided by Takechi et al. [97]. Single o/w or double w/o/w emulsion solvent evaporation method was utilized by Yamamoto et al. [98-100] to prepare microspheres with improved dispersibility by dispersing a w/o type emulsion in an outer aqueous phase that contains an osmotic pressure regulating agent [98] or to prepare sustained release microsphere containing a LHRH derivative or its salt in a large amount without containing gelatin by using a lactic acid-glycolic acid polymer or salts. When the low molecular weight of lactic acid-glycolic acid polymer fraction (8,000 to about 15,000) is contained in a large amount, LHRH derivative readily interacts with these polymers of high reactivity [99], or otherwise to produce a sustained-release composition which comprises emulsifying an aqueous solution containing LHRH derivative and an acid or a base with a solution of a biodegradable polymer [100]. Similarly, Takechi et al. [73] utilized w/o/w or o/w emulsions for encapsulation of thyrotropin-releasing hormone (TRH) and leuprorelin acetate using biodegradable polymers. An improvement in the absorption of a drug that is soluble in an acidic medium (pH less than 3) but slightly soluble in neutral conditions or slightly absorbable and amorphous drugs was provided by Takada et al. [101, 102] where biodegradable microparticles degrade in the digestive tract to gradually release a free acid of water-soluble monomers and oligomers together with the drug substance. Thus, the drug is being solubilized by the released acid, and thus its absorption can be improved. In the process, active drug substance and the polymer are dissolved in an organic solvent. In order to adjust the biodegradation rate of the polymer, an aqueous solution of a pH adjustor may be added, followed by emulsification to prepare a w/o type emulsion that is further added to the third phase (aqueous phase) to form a w/o/w emulsion followed by removal of the solvent by the “inwater drying”. Controlled release gastroretentive microspheres that release an active agent in the stomach environment over a prolonged period of time and can be prepared using a novel spray drying of (w/o/w) and (o/w) or (w/o) emulsions was prepared by Illum and Ping [103]. Microspheres contain an inner core gelling hydrocolloid such as gelatin, a rate controlling layer of a water insoluble polymer such as ethylcellulose and an outer layer of a bioadhesive agent in the form of a cationic polymer such as cationic polysaccharide (chitosan, or diethylaminoethyldextran), a cationic protein, or a synthetic cationic polymer. Insufficient solvent removal, due to the unsatisfactory speed of solvent removal from microspheres, is encountered in the “in-water drying” and is likely to cause sphere 184 Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 Wasfy M. Obeidat aggregation, resulting in problems regarding the decrease in the drug entrapment ratio in the microspheres obtained and the unsatisfactory dispersibility and the needle passability of spheres during administration [72, 73, 100-102]. How-ever, Takechi et al. [104] developed a method to increase the speed of solvent removal from microspheres prepared from a w/o/w emulsion or from an o/w emulsion and markedly improve the drug entrapment ratio in microspheres by subjecting the mirocapsules to an “in water drying” in a tight closed container whose inside is separated from ambient conditions. A gas is allowed to be present above the liquid surface that contains an organic solvent evaporated from the emulsion and which is replaced with an organic solvent-free fresh one by sequential removal of portions at predetermined rates. By optimizing certain conditions such as container size, amount of gas phase and external aqueous phase amount of microspheres in external aqueous phase and replacement frequency and gas transfer rate, then the “in-water drying” method is normally completed in a short time of about 0.5 to about 5 hours. In series of patents, Mesens et al. [105-108] designed microparticles comprised at least one antipsychotic (risperidone) and at least one biocompatible, biodegradable polymer for the controlled extended release of an effective amount of a drug. The organic phase and the aqueous phase are pumped so that they are simultaneously flowing through a static mixer, thereby forming an o/w emulsion which is then pumped through the static mixer into a large volume of quench liquid such as water. In certain processes, solvent evaporation may be replaced by sublimation via freeze drying after the emulsification process. The o/o or w/o/w emulsions are cooled to a temperature which freezes the drug-polymer-solvent but not the continuous phase. The suspension is then subjected to freeze drying permitting the removal of the solvent the continuous phase and that entrapped in the microspheres [109, 110]. Although the process improves the yield of soluble drugs and prevents the hydrolysis of the drug or the polymer, residual solvents pose technological problems. The other way in which the solvent can be removed from the microparticle is through extraction of the solvent present in the internal phase after the emulsification step. This can be achieved by using large volumes of dispersing phase or continuous phase with respect to the dispersed phase. The amount and composition of the continuous phase are chosen so that the entire volume of the disperse phase solvent can be dissolved. Otherwise, the removal of the solvent in this method can be achieved by choosing a dispersed phase consisting of cosolvent, of which at least one a great affinity for the dispersing phase. The method was patented very early in US Patent 3523907 and has been used by many researchers in the field [111]. Other early conventional microencapsulation processes were disclosed in [46, 112] wherein a solution of a wall or shell forming polymeric material in a solvent is that is only partially miscible in water prepared. A solid or core material is dissolved or dispersed in the polymer containing solution and, thereafter, the core material containing solution is dispersed in an aqueous liquid that is immiscible in the organic solvent in order to remove solvent from the microparticles. The removal of the organic solvent is done by extraction with water, however it is limited to certain solvent systems. Several recent patents describe solvent removal by extraction. For example, Cleland et al. [113] discloses the delivery of an adjuvant or an antigen to a host via the use of biodegradable microsphere in a pulsatile or a continuous manner for use in immunization of a patient. The production of microspheres is performed by a double emulsion (w/o/w) to improve the encapsulation using PLGA polymer. However, in the hardening step, the emulsion is transferred to a hardening extraction medium and gently mixed for about 1 to 24 hours to extract the polymer solvent. The long period of time required for extraction is undesirable, particularly if the process is to be operated continuously. Thus, to overcome the drawbacks in long extraction times typically encountered in microspheres dried by vacuum drying or lyophilization (since these methods are time consuming and often result in degradation), Cleland et al. [114] prepared microspheres using an improved w/o/w process; where the second emulsion was performed similar to the first w/o emulsion with 10% PVA and the temperature of the second emulsion was maintained at 0°C to 3°C and hardened by extraction with aqueous medium to provide a final product with less than 20% residual moisture by drying the produced microspheres in a fluidized bed dryer. Solvent extraction is frequently performed as a two-step process. First, the drug-matrix dispersion is mixed with a small amount of continuous phase to yield an emulsion of desired droplet size (distribution). Then, further continuous phase and/or additional extracting agents are added at an amount sufficient to absorb the entire solvent leaching from the solidifying microspheres. Nonetheless, Vuaridel and Orsolini [115, 116] provided a one-step solvent extraction process. Without prior emulsification step, the drug (LHRH acetate)-matrix dispersion is immediately homogenized at 20000 rpm with such a quantity of continuous phase that is capable of dissolving the total amount of the disperse phase solvent at once in one step process. However, these processes require careful settings of the physicochemical parameters during the homogenization step in order to yield homogenous and intact dispersed drug particles. Tice and Gilley [117] provided an emulsion based method for preparing microspheres with high loading (up to 80 wt. %) of highly water soluble drugs that have a high propensity to partition quickly into the continuous phase of the emulsion. Unfavorable thermodynamics (slow and incomplete extraction) is compensated for by using a large excess of extraction medium saturated with polymer solvent to dissolve all of the solvent in the microdroplets rapidly (within 3 minutes). Greater than 20-30% of the solvent is immediately removed. Ramstack et al. [118, 119] provided a novel method for the preparation of biodegradable and biocompatible microparticles containing a biologically active agent such as risperidone, or testosterone dissolved in a blend of at least two substantially non-toxic solvents, free of halogenated hydrocarbons such as benzyl alcohol and ethyl acetate. The blend was dispersed in an aqueous solution to form droplets. The resulting emulsion was then added to an aqueous extraction medium. One of the solvents in the solvent blend will be extracted in the quench step (aqueous solution) more Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 185 quickly than the other solvent. Owing to the high boiling point of the left solvent (benzyl alcohol) which is not easily removed by evaporation in air or other conventional evaporative means, some of the more rapidly extracted solvent can be added to the quench extraction medium prior to addition of the emulsion. Thus, when the emulsion is added to the quench liquid, extraction of the more rapidly extracted solvent is retarded and more of the second, more slowly extracted solvent is removed. In a similar approach, Herbert and Hazrati [120] used an organic phase that is pumped through a static mixer via a gear drive pump and head at a predetermined flow rates into a quench medium of large volume of water for injection to rapidly extract a portion of the solvent from each microdroplet, creating a concentration gradient within each droplet and forming a polymer skin on the surface that advantageously traps active agent, but which disadvantageously slows extraction of the remaining solvent from the center portion of the microdroplet. However, larger volumes of extraction medium may increase process equip-ment and operating costs, as well as the costs associated with recycling of used extraction medium. A method for encapsulating vitamins, food supplements, oil soluble substances at high loading (70 wt.%) by the solvent o/w emulsion extraction technique is provided by Kvitnitsky et al. [121, 122]. Since evaporating the solvent from the dispersion is not applicable for delicate and sensitive compounds and it is not effective, because diffusion of solvent through a hard polymer wall is very slow, water at 10-30 times higher than the whole quantity of the organic solvent is added to the emulsion for extracting the solvent. Dawson and Koppenhagen [123] employed a relatively high nonionic emulsifier concentration (5-15 wt. %) in an emulsion-extraction method particularly applicable to those active agents that are susceptible to thermal degradation at temperatures above room temperature (i.e. 20°C) such as proteins and peptides, such as enzymes, hormones and antigens. Extraction methods have also use by Tice et al. [124], Eyles et al. [125], Gombotz et al. [126] and by Li et al. [127]. A combination of solvent evaporation and extraction was suggested to improve the economic efficiency of the microencapsulation process. After emulsion for-mation, part of the solvent is removed by evaporation then a sufficient quantity of an extraction fluid is added to harden the microspheres. The two-step solvent removal can be performed in a way that extraction is the first step. Tice et al. [49, 51, 52] described the preparation of microcapsules of improved characteristics containing an anti-inflammatory active agent using the o/w emulsion based on the use of two distinct steps of solvent removal rather than in one process step. Solvent removal process starts with solvent evaporation step using reduced pressure that continued until 40 to 60% of the oily organic phase, followed by a washing step where the residual organic solvent is extracted until hardening of the microcapsules. The two-step solvent removal technique results in a microcapsule product of improved quality and containing a higher level of active agent. Gibson et al. [128130] provided solvent extraction in an incremental, or cascade way which involves introducing the extraction phase into the emulsion through a series of feed streams rather than a single feed stream in order to slow and finely control the extraction of the solvent. Otherwise, the solvent is removed by membrane separation, cryogenic extraction, or by twophase solvent extraction. A brief extraction step prior to evaporation minimizes the loss of active agent from the microparticles. Emulsification based process followed by modification of the chemical composition of the dispersed phase after emulsification leading to microsphere formation by coacervation and cross linking is provided by Orly et al. [131]. The modification of the internal phase was achieved by adding to the formed emulsion either a solution of an alkaline substance in an organic liquid miscible with the aqueous phase if it is desired to render the aqueous phase alkaline, or a solution of an acid substance in an organic liquid miscible with the aqueous phase if it is desired to acidify the aqueous phase making it possible to obtain physicochemical conditions favorable to the reaction in the aqueous phase between a polysaccharide carrying esterified carboxyl groups and a substance selected from the group consisting of a polyamino substance, and a polyhydroxylic substance. A novel method to prepare insulin loaded AcHES-PLGA microspheres for sustained release delivery was introduced by Deluca et al. [132]. In this method, acryloyl hydroxyethyl starch (AcHES) hydrogel microparticles are allowed to swell with vortex mixing with insulin/acetic acid. PLGA/methylene chloride polymer phase is added to the swollen AcHES particles to form a dispersion of w/o type (insulin in hydrogel)/(PLGA in methylene chloride). This primary dispersion is emulsified with PVA solution to form w/o/w emulsion before solvent extraction at 4°C using an ice bath. Then the temperature is gradually elevated to 39°C to facilitate the removal of methylene chloride. Lavik et al. [133] utilized polylactide-co-glycolide (PLGA) or from a blend PLGA and poly lactic acid (PLA) where the molecular weight as well as the ratio of LA (which has a longer degradation time) to GA (which has a short degradation time) are optimized in preparing microspheres using the w/o/w emulsion solvent evaporation technique followed by solvent extraction. The two-step (evaporation and extraction) solvent removal method was utilized by Brown et al. [134] to encapsulate pre-fabricated small spherical particles such as insulin particles, by Liggins et al. [135] to prepare high loading (i.e., higher than 50% w/w) of one or more bioactive agents, by Reid et al. [136] to prepare microspheres as carriers of immunogens for oral, eliminating the need of large doses of antigen to achieve sufficient local concentrations in the intestine, by Vaugn et al. [137] to prepare sustained release non-steroidal anti-inflammatory and lidocaine PLGA microspheres, and by Ramtoola [138] to prepare cyclosporin micro and nano-spheres and by others to produce microcapsules containing active ingredients [139143]. 4. PRODUCTION OF SOLID LIPID MICROPARTICLES USING THE EMULSION METHODS Lipids and oily materials and waxes have been utilized similar to polymers to produce solid lipid micro-particles by Gasco [144-146], Westesen and Siekmann [147-149], Domb [150] In these inventions, a lipid component or a mixture of lipid components, which may contain a pharmacologically 186 Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 Wasfy M. Obeidat active substance, is heated to the melting point; separately an aqueous solution containing one or more surfactants and possibly one or more co-surfactants is prepared, and the resulting solution is heated to a temperature equal at least to the melting temperature of the lipid component or mixture of lipid components; this solution is admixed under mild stirring with the lipid component or mixture of lipid components, obtaining a microemulsion; the microemulsion is poured under stirring in water of 2°C to 10°C, obtaining the formation of well-dispersed lipid microspheres; the dispersion is washed with water by filtration. In other procedures [151, 152] lipids were dissolved or dispersed in an organic solvent or otherwise being melted before the formation of the emulsion. Mixtures of lipids and polymers can be also combined to make the matrix of microparticles after the emulsification process [153]. Morgan and Blagdon [154] prepared edible microcapsules which contain a multiplicity of liquid cores. In the process, a w/o emulsion, with the active ingredient dissolved in an inner aqueous phase, is spray cooled, which causes the solidification of the fat phase and the entrapment of the aqueous phase as minute droplets dispersed in a microcapsule. This process, however, leads to very unstable microcapsules from which the water phase migrates from the inner part of the microcapsule to an outer part. This also results in the condensation of the water on the wall of a container. In addition, the release of the active ingredient cannot be controlled in the microcapsules. Coyne et al. [155] provided a microcapsule which comprises a solidified hydrophobic shell matrix selected from the group comprising fats, oils, waxes, resins, emulsifiers or mixtures of these containing encapsulated gelled or cross-linked aqueous beads using hydrocolloids. Thus the active ingredient is double encapsulated in the microcapsules. One advantage of such system is that the release rate of a water soluble active ingredient in a conventionally spray cooled fat matrix microcapsule is usually not controlled by the melting of the fat matrix but rather by the diffusion of water into the microcapsule and subsequent migration of the active ingredient outside the microcapsule. 5. PRODUCTION OF UNIFORM SIZE MICROSPHERES USING THE EMULSION SOLVENT REMOVAL METHODS Processes and apparatus which can be operated continuously to produce microcapsules were disclosed very early [156-161]. Recently, few patent publications were directed towards improving the microsphere products in terms of particle size and particle size distribution. Muranishi et al. [162] produced polylactic acid microspheres having an average particle diameter of about 0.1 to 10 m. The process involves the formation of o/w or o/o emulsion of fine particles with using a propeller stirrer or turbine stirrer followed by the ultrasonic wave and then distilling away the first solvent. Tice et al. [143] introduced a novel method of producing microcapsules having a size of 1 to 10 m. coumarin, a water-insoluble dye or TNP-KLH, a watersoluble antigen are microencapsulated with a nonbiodegradable polymer (polystyrene) or with a biocompatible, biodegradable polymer (PLGA), respectively. Polymers are dissolved in of methylene chloride and emulsified in aqueous medium containing poly(vinyl alcohol) (PVA) solution saturated with methylene chloride resulting in an o/w emulsion that is transferred to a large volume of deionized water and subjected to gradual pressure reduction that allows for the evaporation of the solvent. Fong [163] and Rössling et al. [164] described the process of preparing narrow particle size microspheres of an o/w and w/o/w emulsion solvent evaporation methods respectively. Fong modified the release rate of a core material (such as ketotifen hydrogen fumarate) by the use of an alkaline agent, e.g. sodium hydroxide that is incorporated either in the water or in the oil phase. While Rössling et al. encapsulated at least 85% of peptides, proteins in biodegradable micro-capsules using non-halogenated solvents to eliminate the potential risk of explosion. A nonemulsion solvent extrac-tion process to produce monodispersed particles in the desired size ranges were invented by Lombardo and Natale [165] and by Fulwyler and Hatcher [166]. In these innovations a latex material such as poly (styrene-allyl alcohol) is disposed in a solvent, which in turn is suspended in droplets in a fluid bath. The solvent and bath materials are chosen such that the solvent is slightly soluble in the fluid bath material, but the latex is essentially insoluble in the bath. Thus, as the solvent diffuses into the fluid bath mate-rial, the latex is continuously redistributed and concentrated. When all of the solvent has diffused out of the latex droplets, a solid latex particle remains, the size of which depends on the amount of latex material in the original droplet. A con-tinuous process for preparing o/w emulsions of controlled particle size within a narrow and/or uniform particle size range was provided by Rourke [167]. By introducing a hydrophobic phase continuously and simultaneously with a hydrophilic phase into the zone of high shear mixers such as colloid mill, an emulsion with a high volume of droplets of hydrophobic phase of substantially uniform size can be made. However, the high shear emulsification process in this invention cannot produce a wide range of microsphere average sizes having a narrow size distribution. Amsden and Liggins [168] provided a population of microspheres having uniform size. Polymer solution phase is pumped at a given rate through a stainless steel needle seated at different angles within TEFLON™-coated polyethylene tubing protruding through the tubing wall. PVA solution in reverse osmosis water solution was pumped at a constant rate of through the polyethylene tubing and past the needle. According to the invention, by controlling various parameters of the method; the interfacial tension, needle gauge number, angle of needle orientation and the viscosity of the PVA solution, microspheres size is controlled. Ipponmatsu et al. [169] produced uniform inorganic microspheres by injecting an aqueous solution containing a particle-forming material into an organic solvent through a macromolecular membrane which has pores substantially uniform in pore size and extending in the direction of thickness, and substantially straight through the membrane, and has a hydrophobic surface. The pores in the membrane are formed by either a corpuscular or laser beam. In addition, a number of publications are encountered in the literature utilizing the emulsion solvent removal method and investigating new equipments and techniques to yield Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 187 monodisperse size distributions of microsphere preparations [170-174]. 6. PRODUCTION OF DOUBLE WALL MICROPHERES USING THE EMULSION SOLVENT REMOVAL METHODS Mathiowitz and Langer [175] described a single step method for preparation of multilayer polymeric drug delivery devices using two or three different degradable or non-degradable polymers that are dissolved in a volatile organic solvent but not soluble in each other at a particular concentration. The drug is dispersed or dissolved in the polymer solution. The mixture is suspended in an aqueous solution and stirred, and the solvent is slowly evaporated, creating multi-layer microcapsules (microspheres) with an inner core formed by one polymer and an outer layer of the second polymer. Microspheres are distinguished by extremely uniform dimensioned layers of polymer. Similarly, Wen and Anderson [176] prepared double wall microspheres using two biodegradable polymers by the o/w emulsification solvent extraction process. In addition, double wall microspheres can be prepared where polymers are melted and combined with the substance to be incorporated, then cooled to form layered microspheres Mathiowitz and Langer [177]. Despite the usefulness of these methods in making uniformly layered microspheres or microcapsules for controlled delivery, they are not applicable to hydrophilic polymers that are not soluble in volatile organic solvents. This problem has been solved by Mathiowitz et al. [178-180] where two or more hydrophilic polymers that are soluble in aqueous or organic solvents but not soluble in each other at a particular concentration and temperature and which have a positive spreading coefficient in solution, were used to form multi-layered polymeric microspheres. In another embodiment the two hydrophilic polymers that gel upon a change in temperature or ionically or covalently cross-linked, or cross-linked by heating upon a change in temperature were separately dissolved to form two polymer solutions. However, solvent evaporation might take long (3 days at 90°C) when using aqueous solvents for dissolving these polymers. Mcginity and Iwata [181] produced unique multi-phase polymeric microspheres in which stable and unstable compounds are dispersed using microemulsions. Specifically, the compound is first separately emulsified with a fixed oil, which is then combined with a polymer-in-solvent combination in the presence of an oil dispersion medium that is incompatible with the polymer solvent to form w/o/o emulsion type. Thus, during the solvent evaporation process, the molecular compound is prevented from diffusing into an outer phase of the emulsion system. Related to this subject, but not in the form a traditional o/w/o or w/o/w emulsions, but rather in the form of multi-lamellar, alternating layers of hydrophilic and hydrophobic immiscible-layer microcapsules contained within a thin, semi-permeable outer skin has been produced by Morrison and Mosier [182]. In the microcapsules of the invention, the immiscible phases are distinct and separated according to the surface tension characteristics of the liquids at each interface, hence there is no true emulsion maintained by the surfactant which could be broken. Spherical multilamellar microcapsules formed have advantages for chemoembolization of vascular tumors. In a modification by the same inventors, methods of using these microcapsules for in situ activation of drugs is described, where upon exposure to an appropriate energy source the internal phases mix and the drug is activated in situ [183]. 7. PROTEIN ENCAPSULATION USING EMULSION SOLVENT REMOVAL METHODS THE As is generally known, peptides and proteins represent active ingredients with a pharmacodynamics that is size dependent. However, peptides and proteins are broken down upon oral administration because of their hydrolysis sensitivity in the acidic environment of the stomach, as well as enzymatic degradation in the small intestine, and thus are partially inactivated in the gastrointestinal tract. Suitable formulations that avoid their instability are depot systems in the form of polymer microcapsules or microspheres. They have the advantages that peptides and proteins are protected against rapid inactivation allowing for lower dosages to achieve an effective pharmacologically activity and the undesirable side effects can be reduced. Since almost all protein drugs are short acting, requiring repeated injections to maintain therapeutic efficacy, thus, currently, the main goal in delivery of protein based pharmaceuticals is to develop controlled release formulations that permit longterm delivery ranging from weeks to months from a single administration. Biodegradable polymers are very attractive, especially when their degradation products are known to be biocompatible. Solvent evaporation and solvent extraction methods have been most frequently employed with PLGA, since it is not water-soluble. However, unfortunately, the solvent evaporation and extraction methods have had limited success with a few select therapeutic proteins. For example Gombotz et al. [184] and Shah [185] worked on the encapsulation of proteins using w/o or w/o/w emulsification solvent removal methods. These methods, however, are not suitable for the majority of proteins due to lengthy procedures and difficulty in scale up for mass production. In many cases, contacts between protein and solvent throughout the microsphere matrix may cause denaturation of most protein drugs to be loaded. Because of the problems associated with using chlorinated organic solvents, the solvent evaporation and extraction methods have not been used as a universal method for making microparticles of protein drugs. To avoid the use of chlorinated organic solvents in microsphere formation, methylene chloride has been replaced with less toxic solvents, such as ethyl acetate, Nmethylpyrrolidone, methyl ethyl ketone or acetic acid [186]. However, the microspheres prepared by methylene chloride are spherical and more uniform, while the use of other solvents such as ethyl acetate results in particles which appear to be partly collapsed. The drug encapsulation efficiency reduces significantly compared to the micro-spheres made by methylene chloride according to Herrmann and Bodmeier [34]. This could be due to the high solubility of ethyl acetate in water, leading to the loss of drug. PLGA polymer is then precipitated by adding alcohol as a nonsolvent and water as a hardening agent but still protein drugs are exposed to organic solvents for a prolonged period of time, and the prepared microspheres are prone to aggregation. In addition, the use of water as a hardening agent 188 Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 Wasfy M. Obeidat may not be ideal for water-soluble drugs, including protein drugs that can dissolve and leach out from the microspheres into the water phase. Since protein drugs will be directly in contact with the polymer matrix, protein molecules may adsorb to the solid surface and become denatured. Hyon and Ikada, Hutchinson and Camble et al. [187-189] proposed the use of acetic acid to prepare PLGA microspheres using the emulsion solvent removal methods. Clearly, acetic acid was used mainly to dissolve PLGA and to dissolve/disperse the active ingredient. Microspheres may be made using conventional solvent extraction and evaporation methods utilizing w/o/w or w/o/w emulsions or using the spray drying method. According to Hyon and Ikada, a solution containing both PLGA and an active substance, such as leutenizing hormone releasing hormone (LHRH) dissolved in glacial acetic acid was mixed with 1/10 volume of aqueous solution of LHRH to obtain complete dissolution of the polymer and the active substance. This solution was added drop wise to oil to prepare an emulsion. Microspheres were obtained by removing water and acetic acid by solvent evaporation at elevated temperatures. While Hutchinson and Camble et al. used glacial acetic acid to dissolve PLGA and dissolve/ disperse peptide drug that is then spray dried or freeze-dried to obtain microspheres. Related to the subject but using nonemulsion solvent removal method, a novel method of producing microcapsules containing proteins using the “solvent exchange method” has been invented by Yeo et al. [190]. This method was employed to produce microparticles with only a minute fraction of protein drugs are directly exposed to the polymer solvent, thereby minimizing the potential for protein denaturation. Biodegradable polymer can be dissolved in a hydrophilic organic solvent such as acetic acid, but it becomes phase separated upon only a small decrease in the solvent quality, i.e., reduced mole fraction of the solvent. The hydrophilic solvent mixes with water at the interface between an aqueous core and the polymer solution. This solvent exchange on the surface of the aqueous core brings about a slight decrease of polymer solubility and results in deposition of a polymer coat around the hydrophilic core. Microcapsules are further hardened in a water bath, e.g., one containing a surfactant to prevent aggregation of microcapsules. Although complex proteins might be unstable when formulated into microspheres, Hurtado et al. [191] produced an antigen microspheres having a native conformational epitope, particularly an antigen derived from the G protein of the human respiratory syncytial virus (RSV) encapsulated in poly (D, L-lactide-co-glycolide) copolymers (PLGA) microspheres with controlled biphasic release, thus mimicking a multiple dose schedule vaccination using the w/o/w emulsion solvent extraction-evaporation method. A second antigen could be adsorbed on blank PLGA microspheres and/or antigen loaded PLGA microspheres. Similarly, Alpar et al. [192] produced vaccine containing microparticles using double emulsion solvent evaporation. The biologically active agent (vaccine) suspended or dissolved in an aqueous solution containing polyvinyl alcohol and a phospholipid (lecithin) is emulsified with a biodegradable polymer dissolved in an organic phase. The resultant emulsion is then dropped into a secondary aqueous phase, also containing polyvinyl alcohol and optionally also the phospholipid with vigorous stirring to for a w/o/w emulsion with subsequent organic solvent evaporation. Sustained release preparation of peptides such as LHRH antagonist leuprorelin in the form of microspheres has been described in quite few patent publications. Kamei et al. [193195] used of the o/w, w/o or w/o/w emulsion solvent removal method. Okada et al. [95, 96] produced microcapsule by preparing a primary w/o emulsion comprising an inner aqueous layer containing LHRH antagonist. This w/o emulsion is added an aqueous layer to give a w/o/w emulsion. [95]. Similarly, Yamamoto et al. [72, 100] and Takechi et al. [73] prepared Leuprolide and Interferon microcapsules using w/o/w emulsion whose viscosity is adjusted to about 150 to about 10,000 centipoises. Or emulsifying an aqueous solution containing LHRH derivative and an acid or base with a solution of a biodegradable polymer to produce w/o or w/o/w emulsions. “In-water drying” was conducted by gradually reducing the pressure while stirring. Futo et al. [196] used a relatively large molecular weight (11,000 to about 27,000) lactic acid polymer or its salt to produce microspheres with prolonged release over a long period of time with a suppressed initial excessive release of a watersoluble LHRH derivative via single or double emulsion. Ducrey et al. [197] incorporated LHRH in the form of a water insoluble peptide salt (The LHRH agonist triptorelin pamoate) to provides slow release microparticles made of a copolymer of the PLGA type (at least 75 % of lactic acid) by the emulsion method. A method of encapsulating DNA retaining its ability to induce expression of its coding sequence in a microparticle for oral administration prepared using the w/o/w emulsion and using biodegradable polymers under reduced shear is produced by Jones et al. [198-203]. In addition, Little et al. [204] provided a high throughput method of preparing multiple (at least 10) different microparticle formulations (containing plasmid DNA) in parallel based on the double emulsion/solvent evaporation technique. Eyles et al. [125] used the w/o/w or an o/w/o emulsions to produce biodegradable microparticles that stimulate production of cytokines in a host cell and containing single-stranded ribonucleic acid (ss-RNA) material, a stabilizing agent and a biologically active macromolecule where the outer surface of the microparticle is free from adsorbed molecules. The encapsulation of hormones such as calcitonin for the sustained release delivery has been achieved by Woo et al. [205]. Biodegradable microspheres prepared using o/w emulsion technique and incorporating release-modifying agents and pH-stabilizing agents that resist changes in pH upon the addition of small amounts of acid or alkali such as basic amino acids, such as L-arginine were prepared. According for the disclosure of the invention, sustained release is affected by the unique interplay of the components of the novel microsphere delivery system. Zale et al. [206] utilized solvent extraction in non emulsion processes developed by Gombotz et al. [126] to prepare sustained release of stabilized, non-aggregated, biologically active erythropoietin (EPO) microparticles. A polymer solution, containing EPO particle dispersion, is processed to create droplets using pressure nozzle. Droplets are then frozen by directing the droplets into or near a liquified gas, such as liquid argon and liquid nitrogen to form frozen microdroplets which are then separated from the liquid gas. The frozen microdroplets are then exposed to a liquid non-solvent, such Emulsion Solvent Removal Patents Recent Patents on Drug Delivery & Formulation, 2009, Vol. 3, No. 3 189 as ethanol, or ethanol mixed with hexane or pentane. The solvent in the frozen microdroplets is extracted as a solid and/or liquid into the non-solvent to form biologically active, aggregation-stabilized EPO containing microparticles. Polysaccharides such as starch have been used as a matrix for encapsulation many active ingredients including proteins. Mcdermott et al. [207] used a non-emulsion method the preparation of an antigen containing starch microparticles. The starch is first dissolved in dimethylsulfoxide at an elevated temperature (50°C - 100°C) and then cooled to a lower temperature, particularly to a temperature below about 35°C., without precipitation. The aqueous starch solution containing a proteinaceous material is added drop wise into water-immiscible fluid such as silicone which is capable of forming a w/o emulsion that is then dispersed into an acetone solution with vigorous stirring and collecting the particles formed. However, Reslow et al. [208, 209] utilized starch to encapsulate vaccines using emulsification method. In process, an immunologically active substance (vaccine) is suspended in an aqueous starch solution with an amylopectin content exceeding 85% by weight before being mixed with an aqueous solution of a polymer having the ability of forming a two phase aqueous system. The starch droplets containing the vaccine are allowed to gel into starch particles through the natural capacity of the starch to solidify and the external phase is washed. Encapsulation of nucleotides and growth hormone using simple or double emulsification methods was achieved by Amos [210] and Johnson et al. [211, 212], respectively. Similar to synthetic polymers, such as poly (lactic acid) or polyorthoesters, proteins have also been used to form microparticles or microspheres for drug delivery. Most are cross- linked in solution using glutaraldehyde, or hardened at elevated temperatures. Unfortunately, there are problems with significant loss of biological activity of incorporated materials and lack of controlled size and in vivo degradation rates. Mathiowitz et al. [213-215] produced protein microspheres that are formed by phase separation of simple w/o or double emulsion techniques followed by solvent removal (evaporation or extraction). Proteins (such as zein) that are hydrophobic, biodegradable, and can be modified proteolytically or chemically to endow them with desirable properties were used. The process does not involve the use of temperatures and agents which degrade most labile proteins. Solid zinc insulin, soluble insulin, vasopressin were encapsulated using this invention. Suslick et al. [216] produced surface modified microparticles that possess a novel protein shell, and a surface coating. The protein shell might consist of cross-linked albumin or other proteins with functional moieties for cross linking, while the surface coating comprises polyethylene glycol, a second protein or an antibody. Microparticles are prepared via emulsification followed by protein agglomeration and cross-linking. The surface coating may be covalently-bonded to the crosslinked protein shell or it may be electrostatically adsorbed to the cross-linked protein shell. The surface of the microparticles can be altered to vary the in vivo pharmacokinetics and biodistribution. 8. CURRENT & FUTURE DEVELOPMENTS This review focused on reviewing most recent and when necessary the earliest patents that utilize the emulsion solvent removal methods for encapsulation of drugs and biologically active agents in terms of the methodology, shell or matrix formers, drugs or active agents and the aim of the encapsulation. Despite the novel patents emerging each year for employing the emulsion solvent removal method to produce uniform, rugged and well optimized microparticles with high degree of reproducibility, the large scale production of microspheres is still problematic. In addition, once produced, the microparticle must then be subjected to an extensive washing process to remove the solvent before the microparticle can be used for further purposes. The inefficiencies generated by these disadvantages increase the cost of the resultant microparticle. Moreover, the organic solvent used to form the microemulsion is a hazardous material which presents health and environmental dangers and requires careful storage, handling and disposal. Therefore the years to come should continue to yield innovative ideas including significant improvement of the physicochemical and toxicological properties of the actual formulations on the market. Thus, preparation technologies capable of producing larger amounts of microspheres in a safe, economic, robust and well-controlled manner are therefore required. CONFLICT OF INTEREST No conflict of interest. ACKNOWLEDGEMENT The author would like to acknowledge University of Sharjah (UAE) and Jordan University of Science and Technology (Jordan) for providing substantial efforts in providing financial and logistic support. The author would like to thank Professor James C Price (UGA/USA) for his contributions in revising the contents of the manuscript. 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