HOMOGENIZATION AND HOMOGENIZERS

Venkatesh Naini Barr Laboratories, Inc., Pomona, New York, U.S.A. Shailesh K. Singh Wyeth-Ayerst Research, Peral River, New York, U.S.A.

INTRODUCTION Homogenization encompasses techniques of emulsication of one liquid into another, dispersing solid particles uniformly in a product, and disrupting cell membranes. Traditionally, homogenizers have been used in the pharmaceutical industry for emulsication. However, they are nding increasing applications in the manufacture of liposomes (1), nanosuspensions (2), solid lipid nanoparticles (3), tablet coating dispersions (4), micro-encapsulation (5), and in cell disruption for harvesting therapeutic proteins in cell cultures (6). Pharmaceutical emulsions are generally classied as oil-in-water (o/w) or water-in-oil (w/o) systems, where the rst component represents the dispersed phase, although more complex systems are feasible. The rst step in the process of emulsication involves application of mechanical energy (homogenization) to break up the dispersed phase and form a stable emulsion. Homogenization is also used for particle size reduction in pharmaceutical suspensions. Important factors controlling the formation of pharmaceutical emulsions and dispersions are mechanical and/or formulation related. Mechanical forces during homogenization cause droplet or particle size reduction by shear, turbulence, impact, and cavitation (7). Shear is caused by elongation and subsequent breakup of droplets, due to acceleration of a liquid. Cavitation is caused by an intense pressure drop, leading to formation of vapor bubbles in the liquid, which implode causing shock waves in the uid. This leads to disruption of droplets, particles, and cell membranes. Homogenizers, available from different manufacturers operate using a combination of these forces (8). This review will focus on commonly used homogenizers in the pharmaceutical industry viz. high-pressure homogenizer, rotor-stator homogenizer, microuidizer, and ultrasonic homogenizer.
Encyclopedia of Pharmaceutical Technology Copyright q 2002 by Marcel Dekker, Inc. All rights reserved.

HIGH-PRESSURE HOMOGENIZATION Auguste Gaulin introduced the rst high-pressure homogenizer in 1900 for homogenizing milk (9). The basic high-pressure homogenizer consists of a positive displacement pump attached to a homogenizing valve assembly (Fig. 1). The pump forces liquid into the valve area at a high pressure. As the product is forced through the adjustable gap (D), its velocity increases tremendously with a corresponding decrease in pressure. The emerging product then impinges on the impact ring (C). This sudden change in energy causes increased turbulence, shear, and/or cavitation, resulting in droplet size reduction and uniform dispersion of particles. Highpressure homogenizers are used in emulsication (10 12), preparation of microparticles and nanodispersions (13 16), liposomes (1, 17, 18), and in cell disruption (6, 19). A laboratory scale model of the high-pressure homogenizer is shown in Fig. 2. For emulsion processing a single-stage or two-stage valve assembly can be used, where 10% of the total pressure is applied at the second stage. Another commonly used approach is the multiple-pass homogenization, if a very narrow particle size distribution is needed. This can be achieved by using a series of homogenizers or processing several discrete passes through the same machine. Several factors affect the nal emulsion formulation obtained using high-pressure homogenization. More importantly the level and type of surfactant (11, 12), level of the oily phase, and homogenization process parameters, such as pressure, number of cycles, or discrete passes through the homogenizer, play a signicant role. Pandolfe studied the effect of several of these factors and found that the premix condition (prior to homogenization), emulsier concentration, and energy input by the homogenizer, signicantly affected the quality of the nal emulsion (11). The effect of increasing homogenizing pressure on emulsions with
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Homogenization and Homogenizers

POOR PREMIX WITH 1% EMULSIFIER 2.0 1.8 Mean Diameter, m 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Percent Oil 20 10 5

Fig. 1 Homogenizing valve assembly in a high-pressure homogenizer. (With permission: APV Homogenizer Group, Wilmington, MA.)

1000

2000 3000 4000 Homogenizing Pressure, psi

5000

GOOD PREMIX WITH 1% EMULSIFIER

Mean Diameter, m

different levels of oily phase, in poor premix (turbine stirrer at 1000 rpm) and good premix (prehomogenized at 500 psi) samples, is shown in Fig. 3. Although droplet size reduction is seen at all conditions, more effective

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 1000

Percent Oil 20 10 5

2000 3000 4000 Homogenizing Pressure, psi

5000

Fig. 3 Mean droplet diameter versus homogenizer pressure for emulsions with 1% emulsier and 5%, 10%, or 20% oily phase. (Adapted from Ref. 11.)

Fig. 2 Laboratory scale model of a Gaulin type high-pressure homogenizer. (With permission: APV Homogenizer Group, Wilmington, MA.)

formulations can be obtained by using the lowest amount of oily phase at the highest homogenization pressure with a properly premixed dispersion. It was also concluded that increasing homogenization pressure could effectively reduce the amount of emulsier required in a formulation. Because of its efcient droplet size reduction, high-pressure homogenization can be used for preparing parenteral fat emulsions (10). Here the requirement is that number of droplets or particles above 1 mm should be limited and no particle should be larger than 5 mm. Calvor and Muller (13) used high-pressure homogenization and a novel method to prepare biodegradable microparticles of poly(D ,L -lactide) (PLA) and poly (D ,L -lactide-co-glycolide) (PLGA). They heated the drug polymer-containing suspensions above the glass transition temperature (Tg) of the polymer, followed by high-pressure homogenization. Above the Tg of the

Homogenization and Homogenizers

60 temperature [C] 75 78 79 80

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50

Density distribution q3 Ig (%)

40

30

20

10

0 0.1

1 Particle size (m)

10

100

Fig. 4 Volume size distribution of poly(D ,L -lactide-co-glycolide) particles at a homogenization temperature of 758C (P), 788C (B), 798C (V) and 808C (.). (Adapted from Ref. 13.)

polymer, the viscosity of the dispersed phase was lowered leading to efcient droplet size reduction by homogenization. Fig. 4 shows the effect of homogenization temperature on particle size reduction in PLGA (Tg , 408C) containing systems. Traditionally, Gaulintype homogenizers are known to uniformly disperse and breakup particle agglomerates. Actual breakup of primary particles in the suspension was considered highly unlikely. However, more recently, high-pressure homogenization was successfully employed to prepare nanosuspensions of poorly soluble drugs, starting from micron-sized material (14). Some factors controlling size reduction of drug particles during homogenization include, gap width in the homogenizer, particle shape and size in the feed material, and fragility of drug crystals. Nanosuspensions have promissing applications in injectable formulations of poorly soluble drugs or reformulating solution parenterals, which contain toxicologically less favorable excipients. They can also be used to enhance saturation solubility and bioadhesive properties of drugs in the GIT, leading to better bioavailability following oral administration (2). Due to the abrasive nature of suspended drug particles, erosion of contact surfaces and heavy-metal contamination is a major concern during high-pressure homogenization. A study by Krause and coworkers

found that heavy-metal contamination was minimal (,1 ppm) in nanosuspensions after being homogenized at 1500 bar for 50 cycles (20). Liposomes are phospholipid vesicles containing an aqueous compartment surrounded by one or more bilayers (1). They are nding increasing application as carriers for small molecule drugs, controlled release and targeting of protein and peptide therapeutics, and as immunological adjuvants in vaccines (17). Due to the realization that liposomes need to be produced on a large-scale, highpressure homogenizers are well suited for industrial production under aseptic conditions. The effects of shear and cavitation during homogenization usually form small unilamellar vesicles (SUVs). The number of passages through the homogenizer and pressure used affects the vesicle sizes (17, 18). For a certain combination of lipid and water, increasing homogenization pressure produces smaller and narrower (decreasing polydispersity) vesicles with an optimum diameter. Any further increase in the number of passes results in broader size distributions due to coalescence (17). Bachmann and coworkers, used a continuously operating high-pressure homogenizer to scale-up production of liposomes using the one-step method, where SUVs are prepared from powdered lipid and aqueous drug solution (18). Encapsulation efciency and entrapped aqueous volume of the vesicles are

1482 Table 1

Homogenization and Homogenizers Encapsulation efciency and entrapped volumes of vesicles prepared using a high-pressure homogenizer Homogenizing pressure (70 MPa) Aqueous volume entrapped (l/mol) 0.91 0.80 0.62 0.58 Encapsulation efciency (%) 11.6 9.0 7.5 6.1 Aqueous volume entrapped (l/mol) 0.89 0.69 0.58 0.47

40 MPa Encapsulation efciency (%) 11.8 10.4 8.0 7.6

Number of cycles 1 5 10 20
(From Ref. 18.)

summarized in Table 1. Liposomes prepared with phosphatidylcholine fraction of soybean lecithin (SPC) were homogenized at 40 MPa or 70 MPa, with the higher pressure producing much smaller vesicles. However, extensive recirculation decreased these differences after several passes through the homogenizer. In addition, encapsulation efciencies decreased at higher pressures and repetitive processing (Table 1). Recent advances in biotechnology have produced several new protein drugs from mammalian and bacterial cell cultures. High-pressure homogenization is widely used to harvest intracellular proteins and enzymes of interest in cell cultures (6, 19). Lander et al. conducted a mechanistic study of cell disruption caused by homogenization (19). Shear and cavitation were found to play an important role in cell membrane disruption and release of intracellular contents. High-pressure homogenizers for cell disruption applications use special valve assemblies for efcient rupture of cell walls (6). Other process parameters to consider during cell disruption are viscosity of the cell suspension, ow rate, and number of passes through the homogenizer.

MICROFLUIDIZATION The microuidizer is a high-pressure homogenizer that works, on a different principle. The pre-homogenized liquid is forced through an interaction chamber using a high-pressure pump. The interaction chamber consists of ceramic microchannels, which cause the liquid feed to split into two streams. These streams are then recombined at very high velocities producing forces of shear, impact, and cavitation, which cause droplet or particle-size reduction in emulsions and suspensions. A complete

description of the operation of the microuidizer is summarized in US Patent 4,533,254 (21). A schematic diagram of the microuidizer process is shown in Fig. 5. These homogenizers are commercially available from Microuidics Corporation (Newton, MA). Microuidizers are capable of handling emulsions (21 23), articial blood (24, 25), suspensions (26), and liposomes (27 30). A microuidizer that can operate at process pressures of up to 40,000 psi is shown in Fig. 6. Because of their efcient droplet size reduction and ease of scale-up, microuidizers are frequently used to prepare parenteral feeding emulsions (22, 23). Droplet diameters were directly related to the process pressure used, number of passes through the microuidizer, and concentrations of emulsier and oily phase in the emulsion (22). Reduction in droplet size of a 10% emulsion as a function of number of passes through the microuidizer is shown in Fig. 7 (22). When the homogenizer was operated at its maximum operating pressure of 10,000 psi, droplet diameters decreased from 380 nm for a single pass to a plateau of 250 nm after four cycles, with further processing having no signicant effect. Lidgate et al. used a microuidizer to prepare an o/w parenteral emulsion for use as a vaccine adjuvant and compared its stability to emulsions prepared by other methods (23). Stress tests to induce creaming were used to test emulsions produced by various techniques. Microuidization produced a superior parenteral emulsion compared to a homogenizer mixer. Stability correlated well with increasing number of microuidizer cycles used to process the emulsion (23). The microuidizer has distinct advantages over conventional milling processes in particle size reduction of pharmaceutical suspensions. Absence of heavy-metal contaminants due to surface erosion and easy scale-up to production were observed when using the microuidizer

Homogenization and Homogenizers

Prehomogenized Mixture Homogenized Product

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Outlet Reservoir

Constant Pressure Intensifier Pump Inlet Reservoir

Interaction Chamber

Pressures up to 40000 psi

Pressure Gauge

Fig. 5 Microuidizer processor ow diagram. (Microuidics Corporation, Newton, MA.)

for preparing radiopaque suspensions (25). Several reports deal with scaled up production of liposomes using the microuidizer (28 30). In one study, liposome dispersions with relatively high lipid concentrations (400 mmol/ml) could be processed to narrow size

distributions using the microuidizer (26). In addition, a US Patent (4,776,991) describes the large-scale production of liposome encapsulated hemoglobin for use as a blood substitute (30). Liposomes produced with high-pressure homogenizers usually result in small unilamellar vesicles (SUVs). Their main disadvantages are low encapsulation efciency and tendency to leak their contents more often than multilamellar vesicles (MLVs). Sorgi and Huang used the microuidizer to prepare cationic liposomes, where the active component is not encapsulated, but forms

400

Particle size / nm

300

200 0 1 2 3 4 No. of Passes 5 6

Fig. 6 The M-140K Microuidizer processor. (Microuidics Corporation, Newton, MA.)

Fig. 7 Emulsion droplet size versus number of passes through the microuidizer for a 10% oily phase emulsion processed at 10,000 psi. (Adapted from Ref. 22.)

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Homogenization and Homogenizers

a complex with the liposome using charge interaction (29). The microuidizer was well suited for preparation and scale-up of cationic liposomes of a plasmid DNA, which was successfully used in gene therapy clinical trials (29).

ROTOR-STATOR HOMOGENIZATION The rotor-stator homogenizer is one of the most commonly used pieces of equipment in the pharmaceutical industry. Although they have limited capability in achieving very ne droplets or particles, rotor-stator mixers are capable of handling liquids at much higher viscosities, compared to high-pressure homogenizer and the microuidizer. A rotor-stator homogenizer consists of an impeller in close tolerance to a stationary housing, which restricts the ow of liquid caused by the impeller movement. Shear and impact comminute particles and droplets caught between the rotor and stator (5, 31). The colloid mill is an extreme example of the rotor-stator homogenizer, where the gap between the rotating truncated cone (rotor) and its housing (stator) is adjustable. However, the colloid mill suffers from disadvantages like generation of excessive heat and incorporation of air in the nished product. Various geometries and congurations of the mixing head in the rotor/stator design are available from different manufacturers (8). They can be used in the batch mode and continuous, or in-line, mode. An in-line rotor/stator homogenizer is depicted in Fig. 8. Parameters affecting nal product quality in rotor/stator homogenization are homogenization intensity, residence time of product in the shearing eld, viscosity of the dispersed and continuous phases, surfactant concentration, rotor/stator design,

volume of the mixer, and volume ratio of the two phases (5, 31). Djakovic and coworkers, studied several factors such as homogenization time, emulsier concentration, and homogenization intensity to determine optimal parameters for emulsication, using a rotor-stator homogenizer (32). Mean droplet diameter and polydispersity were used as measures of nal product quality. For constant homogenization intensity (rpm) and mixing time, droplet size and polydispersity decreased with increasing emulsier concentration before reaching an optimum level (32). Maa and Hsu (5) compared the batch mode and a owthrough apparatus, using rotor/stator homogenization for microencapsulation. Emulsion droplets obtained using the ow-through method were consistently higher than the batch mode. Since emulsication is effected by residence time of liquid in the shearing eld, the ow-through method induced lower shear compared to the batch mode. However, using effective recirculation in the ow-through mode can overcome this problem (31).

ULTRASONIC HOMOGENIZATION Sonication emulsies primarily by cavitation. An ultrasonic homogenizer consists of a generator, converter, and horn tip (8, 31). The converter consists of a piezoelectric quartz crystal, which transforms electrical energy into high intensity vibrations and transmits them to the horn tip immersed in the liquid. Droplet size reduction occurs

35

Emulsion droplet size (m)

30 25 20 15 10 5 0 0 10 20 30 40 50 Sonicating Time (sec) 60 70

Fig. 8 An in-line rotor/stator homogenizer. (IKA Works, Wilmington, NC.)

Fig. 9 Effect of sonication time on emulsion droplet size for 0.4 g/ml of poly(methyl methacrylate)/methylene chloride solution in 6% of polyvinyl alcohol (PVA) solution at a volume ratio of 15:2 (ml/ml) sonicated at 20% (W), 50% (A), and 100% (.) of full power. (Adapted from Ref. 31.)

Table 2 Model (manufacturer) Capacity Batch (100 ml) Continuous (11 L/h) Up to 50000 L/h Laboratory scale; batch or continuous Maximum pressure: 30000 psi Mode of operation (Batch/continuous) Operating parameters

List of homogenization euipment spplied by various manufacturers

Type of homogenizer

Applications Emulsions, nanodispersions, liposomes, ointments, cell disruption, vaccines, parenteralemulsions

Homogenization and Homogenizers

High-pressure

APV Model 2000 (APV Homogenizers)

Production scale; batch or continuous 8000 50000 L/h Batch (.60 ml) Continuous (250 600 ml/min)

Microuidizer

Gaulin and Rannie Models (APV Homogenizers) Ariete Model NS8315 (Niro Soavi) M-110Y (Microuidics) Production scale; batch or continuous Laboratory scale; batch or continuous Maximum pressure: 21750 psi Pressure range: 2000 15000 psi Pressure range: 3000 23000 psi

Dispersions, emulsions, cell disruption, encapsulation, liposomes, vaccines, parenteralemulsions

M-140K (Microuidics) Production scale; batch or continuous Production scale; batch Maximum rpm: 3600 Pressure range: 2500 30,000 psi

Laboratory scale; batch or continuous

Pressure range: 8000 40,000 psi

M-210EH (Microuidics)

Rotor-stator

Silverson Model GX25

Batch (1000 ml) Continuous (500 ml/min) Batch (3.8 L) Continuous (5.7 L/min) 2400 L (low viscosity) 400 L (high viscosity) 3500 L/h ,50 ml With booster horn 40 L/min

Emulsions dispersions, pastes, creams, lotions

Ultrasonic Production scale; continuous

Ultra-Turrax UTL (IKA Works, Inc.) Microson XL2007 (Misonix, Inc.) Flocell 800D (Misonix, Inc.)

Production scale; continuous Laboratory scale; batch

Maximum rpm: 6000 Power: 100 W; Frequency: 22.5 kHz Power: 475 W Frequency: 20 kHz

Emulsions, dispersions, cell disruption

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Homogenization and Homogenizers Technology, 2nd Ed.; Gregoriadis, G., Ed.; CRC Press: Boca Raton, 1993; 1, 49 65. 2. Muller, R.H.; Peters, K. Nanosuspensions for the Formulation of Poorly Soluble Drugs I. Preparation by a Size Reduction Technique. Int. J. Pharm. 1998, 160, 229 237. 3. Muller, R.H.; Mehnert, W.; Lucks, J.-S.; Schwarz, C.; Muhlen, A.; Weyhers, H.; Freitas, C.; Ruhl, D. Solid Lipid Nanoparticles (SLN)An Alternative Colloidal Carrier System for Controlled Drug Delivery. Eur. J. Pharm. Biopharm. 1995, 41 (1), 62 69. 4. Bodmeier, R.; Chen, H. Hydrolysis of Cellulose Acetate Butyrate Pseudolatexes Prepared by a Solvent EvaporationMicrouidization Method. Drug Dev. Ind. Pharm. 1993, 19 (5), 521 530. 5. Maa, Y-H.; Hsu, C.C. Liquid Liquid Emulsication by Rotor/Stator Homogenization. J. Control. Release 1996, 38, 219 228. 6. Pandolfe, W.D. Cell Disruption by Homogenization; APV Homogenizers: Wilmington, MA, 1996; 1 20. 7. Walstra, P. Formation of Emulsions. Encyclopedia of Emulsion Technology: Basic Theory; Becher, P., Ed.; Marcel Dekker, Inc.: New York, 1979; 1, 58 128. 8. Scott, R.R. A Practical Guide to Equipment Selection and Operating Techniques. Pharmaceutical Disperse Systems; Lieberman, H.A., Rieger, M.M., Banker, G.S., Eds.; Marcel Dekker, Inc.: New York, 1989; 2, 1 71. 9. Ed. Processing of Emulsions and Dispersions by Homogenization; APV Homogenizers: Wilmington, MA, 1996; 1 23. 10. Bock, T.K.; Lucks, J.-S.; Kleinbudde, P.; Muller, R.H.; Muller, B.W. High Pressure Homogenization of Parenteral Fat EmulsionsInuence of Process Parameters on Emulsion Quality. Eur. J. Pharm. Biopharm. 1994, 40 (3), 157 160. 11. Pandolfe, W.D. Effect of Premix Condition, Surfactant Concentration, and Oil Level on the Formation of Oil-inWater Emulsions by Homogenization. J. Dispersion Sci. Tech. 1995, 16 (7), 633 650. 12. Daniels, R.; Schulz, M.B. Hydroxypropylmethylcellulose (HPMC) As Emulsier for Submicron Emulsions: Inuence of Molecular Weight and Substitution Type on Droplet Size After High-Pressure Homogenization. Eur. J. Pharm. Biopharm. 2000, 49, 231 236. 13. Calvor, A.; Muller, B.W. Production of Microparticles by High-Pressure Homogenization. Pharm. Dev. Tech. 1998, 3 (3), 297305. 14. Grau, M.J.; Kayser, O.; Muller, R.H. Nanosuspensions of Poorly Soluble DrugsReproducibility of Small Scale Production. Int. J. Pharm. 2000, 196, 155 157. 15. Liedtke, S.; Wissing, S.; Muller, R.H.; Mader, K. Inuence of High Pressure Homogenization Equipment on Nanodispersions Characteristics. Int. J. Pharm. 2000, 196, 183 185. 16. Lamprecht, A.; Ubrich, N.; Perez, M.H.; Lehr, C.-M.; Hoffman, M.; Maincent, P. Inuences of Process Parameters on Nanoparticle Preparation Performed by a Double Emulsion Pressure Homogenization Technique. Int. J. Pharm. 2000, 196, 177 182. 17. Brandl, M.; Bachmann, D.; Drechler, M.; Bauer, K.H. Preparation by a New High Pressure Homogenizer Gaulin Micron Lab 40. Drug Dev. Ind. Pharm. 1990, 16 (14), 2167 2191.

mainly by intense shock waves generated in close proximity to the tip. For large-scale applications, ultrasonic homogenizers can be used in the continuous mode with a ow-cell (33). In general, droplet size reduction in ultrasonic homogenizers is affected by sonication intensity, viscosity of the mixture, emulsier concentration, and time of sonication (31, 34). Fig. 9 shows the effect of increasing sonication power on emulsion droplet size in liquid liquid emulsication (31). Higher sonicating power resulted in smaller emulsion droplets. At all power levels the droplet size reduced dramatically, initially followed by a leveling-off phase. Sonication is comparable to rotor-stator homogenization if sufcient power is used. However, as liquid viscosity increases, rotor-stator homogenization is more efcient due to shear effects (31).

EQUIPMENT CONSIDERATIONS A variety of homogenizers capable of performing a range of processes are available. Important considerations during formulation development include the feasibility and availability of pilot and production scale equipment, which can reproduce the same results. A sampling of homogenizers available from selected manufacturers is given in Table 2. High-pressure homogenizers and microuidizers are available in a wide range of capabilities ranging from bench-top models to production equipment capable of handling large amounts of material. These homogenizers are limited in their handling of high viscosity uids compared to rotor-stator homogenizers, which are designed to handle even thick pastes and creams. However, particle size reduction is more efciently carried out using high-pressure homogenizers and microuidizers. Ultrasonic homogenizers are also capable of handling large volumes by using the continuous or ow-through approach. However higher intensities are needed to cause cavitation and droplet reduction in high viscosity uids. As indicated in Table 2, high-pressure homogenizers and microuidizers can be easily adapted for batch or continuous processing. Rotor-stator homogenizers are available either as batch processors or in-line dispersers, which can be used continuously or by recirculating the product.

REFERENCES
1. Brandl, M.M.; Bachmann, D.; Drechsler, M. Liposome Preparation Using High-Pressure Homogenizers. Liposome

Homogenization and Homogenizers 18. 19. 20. Bachmann, D.; Brandl, M.; Gregoriadis, G. Preparation of Liposomes Using a Mini-Lab 8.30 H High-Pressure Homogenizer. Int. J. Pharm. 1993, 91, 69 74. Lander, R.; Manger, W.; Scouloudis, M.; Ku, A.; Davis, C.; Lee, A. Gaulin Homogenization: A Mechanistic Study. Biotechnol. Prog. 2000, 16, 80 85. Krause, K.P.; Kayser, O.; Mader, K.; Gust, R.; Muller, R.H. Heavy Metal Contamination of Nanosuspensions Produced by High-Pressure Homogenization. Int. J. Pharm. 2000, 196, 169 172. Cook, E.J.; Lagace, A.P. Apparatus for Forming Emulsions. US Patent 4,533,254, Aug 6, 1985. Washington, C.; Davis, S.S. The Production of Parenteral Feeding Emulsions by Microuidizer. Int. J. Pharm. 1988, 44, 169 176. Lidgate, D.M.; Fu, R.C.; Fleitman, J.S. Using a Microuidizer to Manufacture Parenteral Emulsions. Biopharm. 1989, 10, 28 33. Zheng, S.; Zheng, Y.; Beissinger, R.L.; Wasan, R.T.; McCormick, D.L. Hemoglobin Multiple Emulsion as an Oxygen Delivery System. Biochim. Biophys. Acta. 1993, 1158, 65 74. Zheng, S.; Beissinger, R.L.; Wasan, D.T.; Sehgal, L.R.; Rosen, A.L. Oxygen Carrying Multiple Emulsions. US Patent 5,438,041, Aug 1 1995. Illig, K.J.; Mueller, R.L.; Ostrander, K.D.; Swanson, J.R. Use of Microuidizer for Preparation of Pharmaceutical Suspensions. Pharm. Tech. 1996, 10, 78 88. 27.

1487 Talsma, H.; Ozer, A.Y.; van Bloois, L.; Crommelin, D.J.A. The Size Reduction of Liposomes with a High Pressure Homogenizer (Microuidizerw). Characterization of Prepared Dispersions and Comparison with Conventional Methods. Drug Dev. Ind. Pharm. 1989, 15 (2), 197 202. Vemuri, S.; Yu, C.; Wangsatorntanakun, V.; Roosdorp, N. Large-Scale Production of Liposomes Using a Microuidizer. Drug Dev. Ind. Pharm. 1990 , 16 (15), 2243 2256. Sorgi, F.L.; Huang, L. Large Scale Production of DC-Chol Cationic Liposomes by Microuidization. Int. J. Pharm. 1996, 144, 131 139. Farmer, M.C.; Beissinger, R.L. Scaled-up Production of Liposome-Encapsulated Hemoglobin. US Patent 4,776, 991, Oct 11, 1988 Maa, Y.-H.; Hsu, C.C. Performance of Sonication and Microuidization for Liquid Liquid Emulsication. Pharm. Dev. Tech. 1999, 4 (2), 233 240. Djakovic, L.M.; Dokic, P.P.; Sefer, I.B. Mathematical and Experimental Essentials of the Emulsication Process: Optimal Parameters Determination. J. Disp. Sci. Tech. 1989, 10 (1), 59 76. Continuous Ultrasonic Processing Cell; Misonix Corporation: Farmingdale, NY, 1998. Higgins, D.M.; Skauen, D.M. Inuence of Power on Quality of Emulsions Prepared by Ultrasound. J. Pharm. Sci. 1972, 61 (10), 1567 1570.

28.

21. 22. 23. 24.

29. 30. 31. 32.

25. 26.

33. 34.