`
`1SE1
`me):
`
`157
`
`165
`
`175
`
`183 L
`
`193 r
`
`203 n
`
`213
`
`217
`
`mbles
`:r 101
`
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`
`15054
`
`- journal of
`controlle
`release
`
`OFFICIAL JOURNAL OF THE CONTROLLED RELEASE SOCIETY
`AND THE JAPANESE SOCIETY OF DRUG DELIVERY SYSTEM
`
`
`
`PHARMACY LIBRARY
`UNNERSWY OF ‘NiSCONSiN
`
`JUL 0 3 2001
`
`Elsevier
`
`Madison, WI 53705
`
`APOTEX 1042, pg. 1
`
`APOTEX 1042, pg. 1
`
`

`

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`
`APOTEX 1042, pg. 2
`
`‘A’—
`J
`
`APOTEX 1042, pg. 2
`
`

`

`Journal of Controlled Release 71 (2001) 339- 350
`
`journal of
`controlled
`release
`
`www .elsevier.com/locate/ jconrel
`
`== Ill > -... Ill
`Q
`Ill z
`Ill
`~
`
`Oil components modulate physical characteristics and function
`of the natural oil emulsions as drug or gene delivery system
`
`Hesson Chung, Tae Woo Kim, Miyun Kwon, Ick Chan Kwon, Seo Young Jeong*
`Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolkok-dong , Sungbuk-ku, Seoul 136-791,
`South Korea
`
`Received 14 September 2000; accepted 24 November 2000
`
`Abstract
`
`Oil-in-water (o/w) type lipid emulsions were formulated by using 18 different natural oils and egg phosphatidylcholine
`(egg PC) to investigate how emulsion particle size and stability change with different oils. Cottonseed, linseed and evening
`primrose oils formed emulsions with very large and unstable particles. Squalene, light mineral oil and jojoba bean oil formed
`table emulsions with small particles. The remaining natural oils formed moderately stable emulsions. Emulsions with
`maller initial particle size were more stable than those with larger particles. The correlation between emulsion size made
`with different oils and two physical properties of the oils was also investigated. The o / w interfacial tension and particle size
`of the emulsion were inversely proportional. The effect of viscosity was less pronounced. To study how the oil component in
`the emulsion modulates the in vitro release characteristics of lipophilic drugs, three different emulsions loaded with two
`different drugs were prepared. Squalene, soybean oil and linseed oil emulsions represented the most, medium and the least
`stable systems, respectively. For the lipophilic drugs, release was the slowest from the most stable squalene emulsion,
`followed by soybean oil and then by linseed oil emulsions. Cationic emulsions were also prepared with the above three
`different oils as gene carriers. In vitro transfection activity was the highest for the most stable squalene emulsion followed by
`oybean oil and then by linseed oil emulsions. Even though the in vitro transfection activity of emulsions were lower than
`the liposome in the absence of serum, the activity of squalene emulsion, for instance, was ca. 30 times higher than that of
`liposome in the presence of 80% (v / v) serum. In conclusion, the choice of oil component in o / w emulsion is important in
`formulating emulsion-based drug or gene delivery systems. © 2001 Elsevier Science B.V All rights reserved.
`
`Keywords: Cationic lipid; Egg phosphatidylcholine; Interfacial tension; Squalene; Stability; Transfection ; Vegetable and animal oils
`
`l. Introduction
`
`--9
`
`Lipid emulsions have been widely used in pharma(cid:173)
`eutical and medical fields as drug carriers. To be
`applied as parenteral, oral or topical formulations,
`
`_*Corresponding author. Tel. : + 82-958-5114-6114; fax: + 82-
`8-5478.
`E-mail address: syjeong@kist.re.kr (S.Y. Jeong).
`
`emulsions must be physically stable and non-toxic
`[1-4]. Since o/w emulsion is a thermodynamically
`unstable system, it is bound to phase-separate with
`time. One of the most important pre-requisites in
`formulating an emulsion, therefore, is to maintain its
`physical stability.· Emulsion size stability is defined
`as the ability to maintain initial particle size dis(cid:173)
`tribution without undergoing phase separation. Many
`factors are known to change the stability of ernul-
`
`Oi68-3659 I 0
`p
`11$ - see front matter © 2001 Elsevier Science B.V. All rights reserved .
`II: S0 168- 3659( 00 ) 00363-l
`
`APOTEX 1042, pg. 3
`
`

`

`340
`
`H. Chung et al. I Journal of Controlled Release 71 (2001) 339- 350
`
`sions. To mention a few, components, composition,
`preparation method and formulation conditions are
`important factors ([1 ,2] and Refs. cited therein). It
`has been a long-standing aim to formulate stable
`emulsions with small particles since the stabilization
`of emulsion could be achieved by particle size
`reduction · [5]. Our aim is
`to produce a stable
`emulsion that has small particles using natural oils.
`We also tried to elucidate what physical or chemical
`factors influence the emulsion size stability. Spe(cid:173)
`cifically, we investigated how different oils affect the
`emulsion particle size and stability. We also investi(cid:173)
`gated whether there is a correlation between emul(cid:173)
`sion stability and initial emulsion particle size and its
`distribution. To this end, 18 different natural oils,
`including vegetable and animal oils, were chosen to
`formulate o/w type emulsions. Many oils that are
`frequently used in producing parenteral emulsions
`have been included in this study.
`There are indications in the literature that the size
`stability of the emulsion changes greatly by changing
`oils [6,7]. The o/w interfacial tension [6,7] or the
`intrinsic viscosity [6] of the oils have a correlation
`with the particle size of the emulsions when binary
`and more complicated oil systems were used. The
`main stabilizing factor, however, is not certain. In
`our dissertation, we also attempted to correlate these
`two physical properties of oils with the stability of
`emulsions.
`We also investigated how the emulsions made
`with different oils could influence the in vitro release
`properties of lipophilic drugs. To this end, squalene,
`soybean oil and linseed oil were chosen to form
`emulsions wherein a lipophilic drug is loaded in the
`discontinuous oil phase. The results show that the in
`vitro release rates of lipophilic drugs are significantly
`different for three emulsion systems.
`Recently, we have reported that cationic o/w lipid
`emulsions can become efficient in vitro and in vivo
`gene carriers [8,9]. We have also demonstrated that
`the transfection activity depends greatly on the
`composition of cationic emulsifiers and co-emul(cid:173)
`sifiers (Submitted for publication). As an on-going
`research project to develop efficient cationic emul(cid:173)
`sion-based gene carriers, we have evaluated whether
`the
`transfection efficiency can be enhanced by
`altering oil components in the cationic emulsion in
`this paper. Our findings demonstrate that emulsion
`
`stability as well as the transfection activity is greatly
`dependent on the choice of the oil component in the
`emulsion.
`
`2. Materials and methods
`
`2.1. Materials
`
`Borage, castor, coconut, corn, cottonseed, evening
`primrose, fish, jojoba bean, lard, linseed, mineral,
`olive, peanut, safflower seed, sesame, soybean, sun.
`flower and wheat germ oils and squalene were
`purchased from Sigma Chemical Company (St
`Louis, MO) and used without further purification.
`L-a-phosphatidylcholine from dried egg-yolk (egg
`PC, 60% pure by TLC) and 2-[(2,6-dichloro(cid:173)
`phenyl)amino]benzeneacetic acid sodium salt (di(cid:173)
`clofenac) were also from Sigma. Rifampicin was
`kindly supplied by Yuhan Pharmaceutical Company,
`Ltd.
`(Korea). 1,2-Dioleoyl-sn-glycero-3-trimethyl(cid:173)
`ammonium-propane (DOTAP) was from Avanti
`Polar Lipids, (Alabaster, AL). Water was purified by
`using a water purification system (Milli-Q Plus;
`Millipore Corp., Bedford, MA).
`
`2.2. Preparation of emulsions
`
`The o I w emulsions contained 100 f..Lll ml of oils
`and various concentrations (1-48 mg/ ml) of emul(cid:173)
`sifiers. In most of the studies, egg PC was used as an
`emulsifier. For in vitro gene delivery experiments,
`DOTAP at 24 mg/ml was used as a cationic
`emulsifier. The emulsifier was weighed and mixed
`with deionized distilled water. The mixture was
`sonicated until clear by using a probe type sonicator
`(High Intensity Ultrasonic Processor, 600 W model,
`Sonics and Materials, Danbury, CT) to form a
`liposome solution in an ice/water bath. The aqueous
`phase was added to oil and sonicated in an ice/water
`bath for ca. 4 min to form emulsions. The emulsions
`were kept at room temperature for further experi(cid:173)
`ments, unless otherwise specified.
`
`2.3. Determination of the particle size of emulsion
`and emulsion/DNA complex
`
`.
`The average particle size of the emulswns as
`
`well
`
`..
`
`I
`I
`
`APOTEX 1042, pg. 4
`
`

`

`H. Chung et al. I Journal of Controlled Release 71 (2001) 339-350
`
`341
`
`tly
`he
`
`mg
`·aJ,
`Jn.
`ere
`(St
`on.
`~gg
`
`1[0-
`:di-
`~as
`ny,
`lyl(cid:173)
`mti
`by
`lus;
`
`oils
`o.ul-
`san
`:nts,
`)nic
`1xed
`was
`a tor
`,del,
`n a
`~ous
`rater
`ions
`Jeri·
`
`~ion
`
`well
`
`as emulsion/DNA complex was determined by
`uasielastic laser light scattering with a Malvern
`ietasizer® (Malvern Instruments Limited, UK).
`Emulsion or emulsion/DNA complex was diluted by
`3oo times before the measurement. The size mea(cid:173)
`surements were performed 1 day after the emulsions
`were prepared and at preset intervals to monitor the
`emulsion size stability. The size determination was
`repeated three times/ sample for at least three sam(cid:173)
`ples comprising an identical composition. It is con(cid:173)
`ventional to show the size distribution function of the
`emulsion as shown in Fig. 1A or B. In this paper,
`however, it would be space consuming to show the
`distribution function of all the emulsions especially
`since we intend to show the size distribution function
`change with time. An alternative way to display data
`would be to show the average size values only
`sacrificing the information on size heterogeneity in a
`single system. To display the data efficiently, we
`adopted a new method to display size distribution
`function data clearly by using the following pro(cid:173)
`cedure in this paper. The size distribution function
`followed a so-called log-normal distribution function
`
`A
`
`8
`
`10
`
`100
`
`1000
`0 200 400 600 800 1 00(
`Size (nm)
`
`1000.0 c
`
`E'
`s 100.0
`
`Q)
`.!::!
`Cf)
`
`10.0
`
`ott
`
`D
`
`300.0
`
`200.0
`
`100.0
`
`0.0
`
`Ij
`
`Fig. l. Typical size distribution of the emulsion function in (A)
`logarithmic axis mode. When the horizontal axis is converted to
`the normal axis mode, the distribution looks positively-skewed as
`shown in (B ). The polydispersity is the variance of the log-normal
`distribution function. From the polydispersity, standard deviation
`can be calculated and is shown as the bars in (C). Therefore, the
`bars in the size distribution function in normal scale (D) are
`calculated from polydispersity in the log-normal size distribution
`function.
`
`for our emulsion systems as shown in Fig. 1 [ 1 0].
`The fact that the size distribution of our emulsion
`systems displays a unimodal log-normal distribution
`has also been confirmed by using a dynamic laser
`light scattering device (Model BI-9000 AT Digital
`Correlator, Brookhaven Instruments Corp., Holts(cid:173)
`ville, NY). Log-normal distribution function shows a
`Gaussian distribution in logarithmic axis mode. The
`variance of the function is called polydispersity. The
`bars in Fig. 1C in logarithmic axis mode indicate the
`standard deviation
`that corresponds
`to
`(poly(cid:173)
`dispersity) 112
`• In the normal axis mode, the dis(cid:173)
`tribution function is positively-skewed (Fig. 1B ).
`The bars in normal axis mode, also positively(cid:173)
`skewed, represent the standard deviation (a') of the
`log-normal size distribution function (Fig. 1D).
`Therefore, in this paper, the average particle size and
`standard deviation will represent the size distribution
`function of an emulsion system.
`
`2.4. Interfacial tension and viscosity measurements
`
`Interfacial tension between oil and aqueous sub(cid:173)
`phase was measured by using du Nouy type surface
`tensiometer (Fisher Surface Tensiomat, Model 21,
`Fisher Scientific Company, Pittsburgh, PA). The
`interfacial tension was measured from more dense
`liquid, aqueous solution to less dense liquid, oil by
`slowly lifting the platinum-iridium ring while in(cid:173)
`creasing the scale to maintain the zero reading. The
`scale reading at the breaking point of the interfacial
`film was the apparent interfacial tension. In this
`paper, apparent values are reported. The measure(cid:173)
`ment was performed at 22±2°C.
`The
`lower and upper phases were deionized
`distilled water and pure oil, respectively, without
`emulsifiers. In some cases, especially for the less
`hydrophobic oils, some of the components diffused
`from the oil phase to the aqueous sub-phase with
`time. We measured the o/w interfacial tension within
`2 min from the contact time between the two phases.
`Therefore, the interfacial tension in this paper does
`not represent the equilibrium values.
`The viscosity of the oils was measured by using a
`kinematic viscometer at 22 ± 2°C (Cannon-Fenske
`Type, Calibrated, Cat. No. 13-617E, Size 200, Fisher
`Scientific, Pittsburgh, PA).
`
`APOTEX 1042, pg. 5
`
`

`

`1
`
`342
`
`H. Chung et al. I Journal of Controlled Release 71 (2001) 339-350
`
`2.5. In vitro release of lipophilic drugs
`
`A lipophilic drug (3 mg) was solubilized com(cid:173)
`pletely in 1 ml oil and mixed with 9 ml of a
`liposome solution of 18 mg I ml egg PC. Linseed oil,
`soybean oil and squalene were chosen as the oil
`phase. The mixture was sonicated in an ice/water
`bath for ca. 4 min to form emulsions. The emulsions
`were kept at room temperature. For the release
`experiment, 2 ml of emulsion was pipetted into a
`dialysis bag (Spectra/Por® membranes, MWCO:
`3500, Spectrum Medical
`Industries,
`Inc., Los
`Angeles, CA). The bag was sealed and immersed in
`10 ml phosphate-buffered saline (PBS) at pH 7.4 at
`room temperature. For the emulsions containing
`rifampicin, ascorbic acid (0.5 mM) was added in
`PBS
`to prevent rifampicin oxidation during the
`release experiment. The tubes were incubated in a
`shaking water bath (Vision Co. Ltd., Korea) at 37°C
`at a shaking frequency of 150 rpm. The release
`medium was exchanged totally with fresh PBS
`solution of an equal volume when the concentration
`of the released drugs was determined. Concentration
`of rifampicin was determined by measuring the
`fluorescence emission (K2 Multifrequency Phase
`Fluorometer, ISS Inc., Champaign, IL) at 480 nm
`("-ex = 370 nm). Before the fluorescence measure(cid:173)
`ments, 2 ml of released medium was reacted with 0.5
`ml each of 0.1 N sodium hydroxide aqueous solution
`and hydrogen peroxide for 2 to 3 h [ 11]. The
`concentration of released diclofenac was determined
`by performing high performance liquid chromatog(cid:173)
`raphy (HPLC) as described elsewhere [12]. Briefly,
`HPLC system consisted of a SP881 0 precision
`isocratic pump (Spectra-Physics Inc., San Jose, CA).
`Mobile phase for diclofenac consisted of 50% (w /w)
`each of acetonitrile and water with a trace of acetic
`acid to adjust pH to 3.3. The flow rate of the mobile
`phase was
`controlled
`to
`1 mllmin. Waters
`f-LBondpack T M C18 Column (3.9 mmX300 mm,
`Waters Corp., Milford, MA) was used. The column
`effluent was monitored at 230 nm by using Spectra
`100 variable wavelength detector (Spectra-Physics).
`
`2.6. In vitro gene transfer
`
`A derivative of simian kidney cell line, COS-I,
`was cultured
`in Dulbecco's modified Eagle's
`
`medium (DMEM, Gibco BRL/Life Technologies,
`New York, NY) supplemented with 10% fetal bovine
`serum (PBS, Gibco) at 37°C in a humidified 5%
`carbon dioxide incubator. Cells were seeded at 2X
`104 cells per well onto 96-well plates 12 h before
`transfection. Cells were ca. 70-80% confluent at the
`time of transfection.
`The plasmid pCMV-beta encoding Escherichia
`coli (E. coli) lacZ ([3-galactosidase) gene expression
`plasmid driven by the human cytomegalovirus imme(cid:173)
`diate-early promoter was purchased from Clontech
`Laboratories (Palo Alto, CA). The plasmid was
`amplified in the E. coli DH5-a strain and purified by
`using a Qiagen mega-kit (Qiagen Inc., Chatsworth,
`CA) according to
`the manufacturer's instruction.
`DNA purity was determined by agarose gel electro(cid:173)
`phoresis and by measuring optical density. DNA
`having OD 260 /0D 280 2:::1.8 was used. pCMV-beta
`(0.5 f-Lg) and 0.0556 f-Ll of emulsion (corresponding
`to 2 f-Lg DOT AP) each diluted with 20 f-Ll serum-free
`DMEM were mixed to form a complex. After
`washing the COS-1 cells with serum-free DMEM, 40
`f-Ll of complex and 160 f-Ll serum free DMEM were
`added to each well. To test effect of serum, 160 f1l
`PBS was added instead of serum free medium. After
`1 h of incubation, the cells were washed with serum(cid:173)
`free media to remove the remaining emulsion/DNA
`complexes. The cells were fed again with DMEM
`containing 10% (v /v) PBS and cultured for 24 h
`after transfection. The transfected cells were assayed
`for [3-galactosidase activity using a photometric
`assay.
`
`3. Results
`
`3.1. Emulsion particle size and size stability
`
`The o/w emulsions with different oils (100 1-111
`ml) were prepared by sonication with 12 mg /ml egg
`PC as an emulsifier in water. The average size of the
`emulsion and standard deviation ( = (polydispe:(cid:173)
`sity) 1 12 in logarithmic axis mode) are summarized ~n
`Table 1. The average sizes of the emulsions were ~n
`s 10
`the range 200-400 nm. The size of the emulsiOn . d
`1 day after preparation at room temperature vane
`depending on oils. Squalene formed the smalle~~
`emulsions (190 nm, polydispersity 0.1 3). Jojoba 01
`
`0
`
`1
`
`I
`
`APOTEX 1042, pg. 6
`
`

`

`·> a=
`Ill > -~
`Ill
`Q
`Ill z
`Ill
`c:J
`
`H. Chung et al. I Journal of Controlled Release 71 (2001) 339-350
`
`343
`
`'
`
`1
`~~leaverage particle size and polydispersity of natural oil emulsions and the o/w interfacial tension (22±2°C) and viscosity of oils
`(20:!:2oc) _ _ _________________ _____________________ _
`~
`Size (nm)
`Polydispersity
`Surface tension
`Viscosity
`Oil
`(dyne/em)
`(eSt/ s)
`
`castor oil
`I coconut 01
`)
`'1
`corn oil
`.
`Cottonseed oil
`Evening primrose ml
`Fi h oil
`Jojoba oil
`Lard oil
`Linseed oil
`Olive oil
`Peanut oil
`Safflower oil
`Sesame oil
`Soybean oil
`Squalene
`Sunflower oil
`Wheatgerm oil
`Mineral oil
`
`273.8
`246.3
`261.0
`263.6
`247.1
`247.0
`224.7
`282.7
`354.9
`263.2
`256.8
`283.6
`263 .3
`249.7
`191.7
`249.2
`253.2
`209.9
`
`0.306
`0.216
`0.220
`0.199
`0.257
`0.251
`0.213
`0.271
`0.116
`0.195
`0.212
`0.225
`0.246
`0.279
`0.125
`0.185
`0.217
`0.154
`
`12.8
`
`22.0
`23 .8
`
`15.6
`25 .8
`18.6
`1.8
`23 .2
`23 .3
`22.0
`26.0
`14.0
`33.9
`26.5
`24.0
`
`723.0
`
`70.4
`62.3
`
`42.0
`43.0
`73.4
`51.2
`84.0
`66.1
`54.2
`62.9
`69.3
`15.9
`47.4
`63.1
`37.8
`
`and light mineral oil yielded the emulsions with
`average sizes of 225 nm (polydispersity 0.21) and
`210 nm (polydispersity 0.15), respectively. Castor,
`safflower and lard oils formed the emulsions in the
`size range 270-285 nm. The emulsion with the
`biggest particles was formed by linseed oil (355 nm,
`polydispersity 0.12). The rest of the oils formed the
`emulsions in the size range 240-270 nm.
`The size and distribution of the emulsions were
`monitored for 20 days at room temperature to
`investigate the size stability of the emulsions. Size
`change as a function of time for six representative
`emulsions is shown in Fig. 2. The emulsions were
`kept at room temperature for the first 10 days after
`preparation. After day 10, a portion of each emulsion
`was transferred to a 4 oc refrigerator for another 10
`days. The rest of the emulsion was kept further at
`room temperature for an additional 10 days. The
`sizes of the emulsions with cottonseed, evening
`primrose and linseed oils grew further ca. 10 days
`after preparation when stored at room temperature.
`!he size of these emulsions did not vary significantly
`If they were stored at 4°C after the first 10-day
`.torage period at room temperature. In the case of
`hnseed oil emulsion, phase separation was observed
`by day 20. The sizes of the squalene and jojoba oil
`
`emulsions did not change with time for the first 20
`days beyond the statistical error range. The castor
`and lard oil emulsions were stable at 4 °C, but the
`polydispersity became slightly bigger (0.3-0.4) with
`time at room temperature (data not shown). The
`coconut, corn, fish, olive, peanut, safflower, sesame,
`sunflower, wheatgerm and light mineral oil emul(cid:173)
`sions stayed stable for 20 days at both temperatures.
`In general, the emulsion whose initial size was
`bigger became unstable more rapidly whereas those
`with smaller initial size remained stable for the
`duration of the experiments. Also, the size variation
`of the emulsions was less at 4°C than at room
`temperature. We also have performed preliminary
`experiments on the stability of the squalene, soybean
`oil and linseed oil emulsions at 37, 100 and 120°C.
`The results show that the linseed oil emulsion was
`unstable at these three temperatures. Phase sepa(cid:173)
`ration of the linseed oil emulsion was observed in ca.
`5 days at 37°C and in 5 min at 100 and 120°C. The
`soybean oil emulsion was stable for 20 days at 37°C
`without any size change. At 100 and 120°C, how(cid:173)
`ever, the particle size became bigger ( 400 nm) in 10
`min, but phase separation was not observed. The
`squalene emulsion stayed stable for 20 days at 37°C
`and for 20 min at 100 and 120°C. We also confirmed
`
`APOTEX 1042, pg. 7
`
`

`

`344
`
`H. Chung et al. I Journal of Controlled Release 71 (2001) 339-350
`
`Lineseed Oil
`
`
`
`:::: Wd ]Soybean oil
`
`500
`
`0
`
`0
`
`. ... +
`
`500
`
`10
`
`20
`
`'
`
`0
`
`0
`
`10
`
`20
`
`Evening Primrose Oil
`
`Jojoba bean Oil
`
`:::: ~T i .... ; ..... ~~ I c. c.
`:::ull ::::f j
`
`50:~ 50:~
`
`particles, for comparison. For these three d1fferem : rog/ rr
`systems, we prepared emulsions at three different ! where
`egg PC concentrations, 3, 12 and 30 mg I ml, to :Size ,
`observe the emulsion stability at different emulsifier broad
`concentrations by monitoring the average particle howe'
`size change for 20 days (Fig. 3 ). When the egg PC soy be
`concentration was 3 mg I ml, the average particle roent.
`sizes of the linseed, soybean, squalene emulsions appar'
`were 64 7, 440 and 3 24.6 nm, respectively, imrnedi· co nee
`ately after preparation. Particle size of the linseed oil soybe
`emulsion was the biggest among the three different enhan
`emulsion systems for 10 days, and phase separation obser
`was observed in 20 days. The soybean oil emulsion size c
`was medium in size among the three and the size given
`highe
`distribution became broad in a few days after
`preparation. In 20 days, phase separation was also Amm
`the n
`observed in soybean oil emulsion. Squalene ernul·
`sion had the smallest particle size and narrow size
`ernul~
`distribution for 20 days. At 12 mglml egg PC, the
`3.3.
`average particle size of the three emulsions wa
`much smaller with improved size stability than at 3 the o
`
`-E
`c -Q)
`
`N
`U5
`
`. ,
`·~
`
`•
`
`i
`
`Linseed Oil
`
`Soybean Oil
`
`Squalene
`3 mg/ml
`
`~ II
`
`•
`
`•
`
`• _L_i
`
`c
`0
`
`c
`0
`
`c
`
`l
`l
`. 1 1 l
`"
`"
`l ~
`i
`"' "' .c
`•
`c.
`1 3 6 10 20 1 3 6 1 0 20 1 3 6 10 20 -12 mg/ml
`.L • ~ l ~ ... ·~
`0 l
`" "' .c •
`
`3 6 1 0 20 1 3 6 1 0 20 1 3 6 10 2
`~----------~~~~~~~30~~-gtml
`
`Th
`of tht
`the e
`addr~
`the i
`two
`contr
`serve
`hav
`viscG
`these
`ernul
`L'
`Whe1
`agai1
`inter
`
`8
`
`6
`
`4
`
`2
`
`0
`
`4
`
`2
`
`0
`
`2
`
`..-
`E
`
`::t -Q)
`
`N
`U5
`
`Time (day)
`ibuuon of
`·
`.
`.
`.
`;mi.
`Fig. 3. Changes in the average particle size and Its distr
`I ·
`at 3 mg
`the linseed oil, soybean oil and squalene emu s10ns
`12 mg/ml and 30 mg/ml of egg PC concentrations.
`
`0
`
`10
`
`20
`
`Cottonseed Oil
`
`1000
`
`1500~··········
`
`500
`
`0
`0
`
`Squalene
`
`0
`
`10
`
`20
`
`::::l'l
`50:~~ dJ
`
`10
`
`20
`
`0
`
`10
`
`20
`
`Time (day)
`
`Fig. 2. Time-dependent changes in average particle size and its
`distribution of emulsions prepared with different oils. Egg PC ( 12
`mg/ml) was used as an emulsifier. The emulsions were kept at
`room temperature for the first 10 days. At day I 0, a portion of
`each emulsion was transferred to and kept in a 4°C refrigerator for
`the rest of the experiment (open circles). The rest of the emulsion
`was stored further at room temperature (filled circles).
`
`that squalene emulsion could be autoclaved without
`sacrificing the emulsion stability.
`
`3.2. Dependence of emulsion stability on emulsifier
`concentration and choice of oil
`
`In forming an emulsion, it is essential to add an
`appropriate amount of emulsifier into oil and water
`to stabilize the system. The concentration of the
`emulsifier is one of the important factors in de(cid:173)
`termining the emulsion size stability. Among the
`emulsions prepared with different oils, squalene
`emulsion had the smallest average size while the
`linseed oil emulsion had the biggest (Table 1 and
`Fig. 2). Therefore, we chose these two systems to
`investigate the correlation between emulsifier con(cid:173)
`centration and particle size in the emulsion. We also
`selected soybean oil, which forms medium-sized
`
`APOTEX 1042, pg. 8
`
`

`

`H. Chung et al. I Journal of Controlled Release 71 (2001) 339-350
`
`345
`
`>(cid:173)It:
`Ill > -... Ill
`
`Q
`Ill z
`Ill "
`
`7 4o ,_8___,.-.,.-----.,--,----.
`•
`
`720
`
`(i)
`'IV
`:;:::. 100
`(j)
`
`~ c 80
`
`'U)
`0
`(.)
`
`(/) >
`
`•
`
`60
`
`40
`
`•
`
`•
`
`• •• •
`
`35 A •
`
`30
`
`E' (.) -Q)
`
`20
`
`15
`
`10
`
`5
`
`c
`>-
`25
`~
`c
`0
`' U)
`c
`._.
`Q)
`cti
`'(3
`ct!
`'t:
`Q) c
`
`••• • ••
`•
`• • •
`
`•
`
`;rnl egg PC. Linseed oil emulsion was unstable
`1erent 1
`'f
`mg eas squalene emulsion was stable for 20 days.
`her
`eren1
`11
`inl 1
`: distribution of soybean oil emulsion became
`0
`,ze
`1'
`d
`'1
`U l'k
`1 ·
`'
`d in 20 days.
`n I e msee
`emu sion,
`tlsifie
`01
`b d'
`r ) broa
`.
`ever phase separatiOn was not o serve m
`artie]
`e hOW
`.
`'
`.
`.
`?,g Pc
`bean oil emulsiOn for the duratiOn of the expen-
`' ;~nt. Squalene emulsion stayed stable with. no
`article
`lsions
`arent size changes for 20 days. At the emulsifier
`ap~centration of 30 mg I ml, size stability of the
`tmecti.
`0
`.
`:ed oil oybean oil and squalene emulsiOns . was greatly
`ferent enhanced. Even though phase separatiOn was not
`[ation observed for linseed oil emulsion for 20 days, the
`ulsion ( ize distribution became b:oader with time. ~or any
`e size j oiven oil systems, emulsiOn has smaller size and
`after
`hiuher stability at higher emulsifier concentrations.
`A~ong the three emulsions, squalene emulsion was
`s also
`the most stable with small particles at any given
`emu].
`v size emulsifier concentrations.
`2, the
`~ was 3.3. Oil/water inteifacial tension and viscosity of
`n at 3 the oil
`
`There are many physical and chemical properties
`of the oils that could regulate the size and stability of
`the emulsions. Recently, Jumaa and co-workers have
`addressed that the olw interfacial tension [6,7] and
`the intrinsic viscosity [7] of the oils are considered
`two of the most important physical properties that
`control emulsion particle size. To test if their ob(cid:173)
`ervation could be reproduced in our oil systems, we
`have determined the oil/ water interfacial tension and
`viscosity of the oils and the relationship between
`these physical properties and the particle size of the
`emulsions (Table 1 ).
`Linseed oil had the lowest interfacial tension
`whereas squalene had the highest interfacial tension
`against water among the studied oils. The olw
`interfacial tension was plotted against the size of the
`emulsion made of that particular oil by using egg PC
`as an emulsifier (12 mglml) in Fig. 4A. We note that
`o/w interfacial tension represents the values without
`emulsifiers. The interfacial tension and the particle
`ize of the emulsion, immediately after preparation,
`Were inversely proportional. When the o I w interfa(cid:173)
`cial tension was the biggest, as in squalene, the size
`of the emulsion was the smallest. The interfacial
`tension was the smallest for linseed oil that produced
`~e emulsion with the biggest partiCle size. Since
`
`g/ml
`
`10 20
`
`lg/ml
`
`Jtion of
`mgfml,
`
`20
`0
`• ''---'---L_..J....____J
`150 200 250 300 350 400
`150 200 250 300 350 400
`Size (nm)
`
`Fig. 4. Correlation between the emulsion size and (A) the o/w
`interfacial tension and (B) viscosity. The emulsions were com(cid:173)
`posed of natural oils (100 f.d/ml) and egg PC (12 mg/ml). The
`interfacial tension was measured between water and pure natural
`oil that was used to form the corresponding emulsion at 22±2°C.
`Viscosity of pure oils were measured at 20±2°C.
`
`more hydrophobic oil has a larger oil/ water interfa(cid:173)
`cial tension, it is possible to infer that more hydro(cid:173)
`phobic oil forms emulsion with smaller particles
`when egg PC is used as an emulsifier.
`Similarly, correlation between viscosity of oils and
`the particle size of the emulsion was evaluated.
`Castor oil is one of the most viscous oils (Table 1 ).
`Squalene was the least viscous among the studied
`oils. The viscosity of the oils was plotted against the
`size of the emulsion containing egg PC as an
`emulsifier (12 mglml) in Fig. 4B. In general, no
`correlation between viscosity of the oils and size of
`the emulsions was apparent. In the case of the
`linseed oil emulsion, the emulsion particle size was
`the biggest among
`the
`investigated oil systems
`although the viscosity of linseed oil was smaller than
`corn or olive oil. The viscosity of castor oil was
`m