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Cyclodextrins as solubilizers: Formation of complex aggregates
Cyclodextrins as Solubilizers: Formation of Complex Aggregates PHATSAWEE JANSOOK, SERGEY V. KURKOV, THORSTEINN LOFTSSON Faculty of Pharmaceutical Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland
Received 5 March 2009; revised 19 May 2009; accepted 20 May 2009 Published online 7 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21861
ABSTRACT: Three different techniques were applied to investigate aggregation of drug/ cyclodextrin complexes, that is, drug permeation through semi-permeable membranes, determination of changes in the value of activity coefficients of drug/cyclodextrin complex solutions and transmission electron microscopy (TEM). The aqueous solutions studied contained g-cyclodextrin, 2-hydroxypropyl-g-cyclodextrin or mixtures thereof, and hydrocortisone, amphotericin B, diclofenac sodium or indomethacin. The permeation studies indicated that drug/cyclodextrin complex monomers (i.e., unaggregated complexes) were dominating at cyclodextrin concentrations below 5% (w/v). Then formation of aggregates gradually increased with increasing cyclodextrin concentration until all increase in dissolved drug/cyclodextrin complexes was due to formation of cyclodextrin aggregates. This happened even though the observed phase-solubility diagrams were linear, that is, were of AL-type. The activity coefficients showed positive deviation from ideal state. This positive deviation is due to concurrent of several processes, that is, hydration, aggregation and complex formation. The observed deviations from ideality indicated that complex aggregates were formed in the aqueous complexation media. TEM pictures showed formation of aggregates in both pure aqueous cyclodextrin solutions as well as in cyclodextrin solutions that had been saturated with hydrocortisone. The aggregate diameter was between 10 and 100 nm but larger aggregates with diameter of about 200 nm were formed through assemble of smaller aggregates. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:719–729, 2010
Keywords:
cyclodextrin; complex; aggregate; permeation; activity coefficient; TEM
INTRODUCTION Cyclodextrins are hydrophilic cyclic oligosaccharides with a lipophilic central cavity. In aqueous solutions cyclodextrins are able to solubilize many hydrophobic drugs by taking up some lipophilic moiety of the drugs into the cavity, that is, through formation of water-soluble inclusion complexes. Aqueous cyclodextrin solutions are frequently used as nontoxic solvents during screening of new chemical entities (NCE) both Correspondence to: Thorsteinn Loftsson (Telephone: þ354525-4464; Fax: þ354-525-4071; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 719–729 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
in vitro and in vivo, and cyclodextrins can be found in just over 30 marketed drug products worldwide.1 In aqueous solutions free drug and cyclodextrin molecules are in dynamic equilibrium with the complexes and every complex unity is generally assumed to be free and independent of other complexes as well as of other excipients found in the solution.2 It is however becoming increasingly apparent that such assumptions may not be universally applicable or all encompassing. Specifically, there is a growing body of evidence that supports the important contribution of noninclusion aspects for drug solubilization by cyclodextrins including surfactant-like effects and molecular aggregation, that is, formation of particulate systems.3–10 It is
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known that spontaneous opalescence occurs in aqueous g-cyclodextrin (gCD) solutions during storage11 and investigations of aqueous 1% (w/v) gCD solutions have shown that about 0.02% (w/w) of gCD in the solutions is in the form of aggregates.12 Comparable observations have been made in a-cyclodextrin (aCD) and b-cyclodextrin (bCD) solutions.13,14 The diameter of these cyclodextrin aggregates is approximately 100 nm. The cyclodextrin derivatives, such as 2-hydroxypropyl-b-cyclodextrin (HPbCD), are also thought to form aggregates in aqueous solutions.8 In aqueous solutions the relative amount of aggregated cyclodextrin increases with increasing cyclodextrin concentration and permeation studies of hydrocortisone/HPbCD solutions have indicated that in spite of the transparent nature of the solutions virtually all increase in drug solubility observed at HPbCD concentrations above 10% (w/v) is due to hydrocortisone/HPbCD aggregate formation.8,15,16 However, aggregates of the natural aCD, bCD, and gCD were studied in very dilute (about 1%, w/ v) aqueous cyclodextrin solutions and the hydrocortisone/HPbCD aggregate formation was only observed by permeation studies. In this present study three different techniques are used to verify formation of the aggregates. Water-soluble polymers, metal ions and various carboxylic acids are also known to influence the cyclodextrin solubilization of drugs, possibly through interactions with cyclodextrin aggregates.17–19 Previously we have shown that addition of relatively small amounts of the water-soluble 2-hydroxypropyl-g-cyclodextrin (HPgCD) to aqueous gCD formulations increases the complexation efficiency (CE) of gCD and reduces the turbidity of gCD solutions. Mixing HPgCD with gCD resulted in synergistic effect with regard to drug solubilization in aqueous media.20 It was hypothesized that the observed effect was somehow related to formation of gCD and HPgCD nanoparticles. The purpose of this present study was to investigate further the solubilizing effects of gCD, HPgCD, and gCD/HPgCD mixtures and their formations of nanosize aggregates.
MATERIALS AND METHODS Materials Hydrocortisone (HC) was purchased from ICN Biomedicals (Aurora, OH), amphotericin B (AmB), JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
diclofenac sodium (DC-Na) and indomethacin (IDM) from Sigma (St. Louis, MO), g-cyclodextrin (gCD) and 2-hydroxypropyl-g-cyclodextrin (HPgCD) MS 0.6 (MW 1576 Da) from Wacker Chemie (Munich, Germany), disodium edetate dehydrate (EDTA) and sodium chloride from Merck (Darmstadt, Germany), and benzalkonium chloride from Sigma. Semi-permeable cellophane membranes (SpectaPor1, molecular weight cut-off (MWCO) 3500, 6–8000, 12–14,000) was purchased from Spectrum Europe (Breda, Netherlands). All other chemicals used were of analytical reagent grade purity. Milli-Q (Millipore, Billerica, MA) water was used for the preparation of all solutions.
Phase-Solubility Profiles The solubility of the drugs was determined by previously described heating method.21 Hydrocortisone, diclofenac sodium and indomethacin were chemically stable (i.e., their degradation was less than 1%) during heating (1218C for 20 min) in aqueous gCD and HPgCD solutions. However amphotericin B was chemically unstable during heating in an autoclave and thus amphotericin B solutions were saturated through heating in a sonicator to 608C for 30 min. The suspension was heated to promote drug, and in some case cyclodextrin, saturation of the eye drop complexation medium. Excess amount of the drug to be tested was added to a solution containing 0–20% (w/v) cyclodextrin in eye drop vehicle consisting of aqueous solution containing 0–20% (w/v) cyclodextrin, benzalkonium chloride (0.02%, w/v), EDTA (0.1%, w/v) and sufficient sodium chloride to obtain isotonicity. The drug suspensions were heated in an autoclave (1218C, 20 min), or in the case of amphotericin B to 608C for 30 min in a sonicator, and allowed to cool to room temperature. Then a small amount of solid drug was added to the suspensions to promote drug precipitation and the pH adjusted to 7.4 with concentrated sodium hydroxide solution. The pH was periodically tested during the equilibration time and the samples were ready to be analyzed. The suspensions were equilibrated at room temperature (22–238C) for 7 days under constant agitation. Preliminary experiments showed that 7 days are more than enough time to reach solubility equilibrium. After equilibrium was attained, the suspensions were filtered through 0.45 mm cellulose membrane filter, the filtrates diluted with the mobile phase and analyzed by HPLC. The DOI 10.1002/jps
CYCLODEXTRINS AS SOLUBILIZERS: FORMATION OF COMPLEX AGGREGATES
apparent complexation constants for drug/cyclodextrin complexes (K1:1) were determined according to the phase-solubility method of Higuchi and Connors.22 The higher order complexation constants were determined through nonlinear fitting of the AP-type phase-solubility diagrams.22–24 The complexation efficiency (CE) was determined from the slope of linear phase-solubility diagrams (plots of the total drug solubility ([drug]t) versus total cyclodextrin concentration ([CD]t) in moles per liter):21 CE ¼
Slope ½drug=CD complex ¼ 1 Slope ½CD
¼ K1:1 S0
(1)
where K1:1 is the stability constant of the drug/ cyclodextrin 1:1 complex and S0 is the intrinsic solubility of the drug.
Quantitative Determinations The quantitative determinations of the individual drugs were performed on a reversed-phase HPLC component system from Hewlett Packard Series 1100, consisting of a G132A binary pump with a G1379A solvent degasser, a G13658 multiple wavelength detector, a G1313A auto sampler, and Phenomenex Luna 5 mm C18 reverse-phase column (150 mm 4.6 mm). The HPLC chromatographic conditions were shown in Table 1.
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membrane. Before usage the membrane was soaked overnight in the receptor phase that consisted of pH 7.4 phosphate buffer saline containing 5% (w/v) mixture of gCD/HPgCD (50/ 50 weight mixtures). To avoid possibility of membrane fouling the receptor phase was autoclaved at 1218C for 20 min prior to use. Cyclodextrin was added to the receptor phase to ensure sufficient drug solubility to maintain sink condition throughout the experiment. The receptor phase was sonicated under vacuum to remove dissolved air before it was placed in the receptor chamber. The donor phase (2 mL) consisted of 0–20% (w/v) cyclodextrin solution in eye drop vehicle saturated with the drug, as previously described under methods in the phase-solubility section. The study was conducted at room temperature (22–238C) and under continuous stirring for 6 h by a magnetic stirring bar rotating at 300 rpm. An aliquot of receptor medium (150 mL) was withdrawn at 30, 60, 120, 180, 240, and 360 min and replaced immediately with an equal volume of fresh receptor phase. The drug concentration in the receptor phase was determined by HPLC. The steady state flux was calculated as the slope (dq/dt) of linear section of the amount of drug in the receptor chamber (q) versus time (t) profiles, and the apparent permeability coefficient ( Papp) was calculated from the flux (J) according to Eq. (2): J¼
dq ¼ Papp Cd A dt
(2)
where A is the surface area of the mounted membrane (1.77 cm2) and Cd is the initial concentration of the drug in the donor phase.
Permeation Studies The permeability of individual drugs was carried out using Franz diffusion cell apparatus consisted of a donor and a receptor compartment (FDC 400 15FF, Vangard International, Neptune, NJ). The donor chamber and the receptor compartment were separated by a semi-permeable cellophane
Determination of Activity Coefficients The concentration dependences of osmolalities were measured using Knauer K-7000 vapor pressure osmometer (Knauer, Germany). The
Table 1. HPLC Conditions Drugs Hydrocortisone Diclofenac sodium Indomethacin Amphotericin B
Mobile Phasea ACN:THF:water (33:1:66) ACN:1.0% acetic acid (60:40) ACN:0.5% acetic acid (50:50) ACN:0.25 mM EDTA (37:63)
Flow Rate (mL/min) Wavelength (nm) Retention Time (min) 1.5 1.5 1.5 1.0
241 282 240 403
5.1 4.0 6.9 3.2
ACN, acetonitrile; THF, tetrahydrofuran; acetic acid, aqueous acetic acid solution; EDTA, aqueous disodium edetate dehydrate solution. a Volume ratios. DOI 10.1002/jps
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osmometer was calibrated with standard sodium chloride solutions of known osmolality. The osmotic coefficients, w, were calculated from osmolalities by equation: Osm ¼ n’m
(3)
where Osm is osmolality of a solute in osmol/kg, n is the number of particles in which the solute molecules dissociate (i.e., n ¼ 1 when there is no dissociation, like in our case), and m is the molal concentration of a solute. The activity coefficients, g, of solutes in the solutions studied were estimated using Bjerrum equation: Zm ln g ¼ ’ 1 þ ð’ 1Þ d ln m (4) 0
The concentration dependency of osmotic coefficients is described by appropriate polynomial function, that is: ’ ¼ 1 þ B1 m þ B2 m2 þ B3 m3 þ
(5)
Then, Eq. (5) is introduced into Eq. (4) instead of w, which results in: 3 4 ln g ¼ 2B1 m þ B2 m2 þ B3 m3 þ 2 3
(6)
As follows from Eqs. (5) and (6) the standard state is represented by an ideal solution with m ! 0, for which both w and g ! 1.
Transmission Electron Microscopy (TEM) Analysis The morphology and size of the aggregates in hydrocortisone saturated aqueous 10% (w/v) gCD, HPgCD and gCD/HPgCD (80/20 and 20/80 weight mixtures) solutions were analyzed using a transmission electron microscope. One of the easiest ways of preparing the samples is by negative staining. This preparation method is useful for visualizing suspensions of small particles. The aggregates were visualized by TEM using the uranyl staining method.25 However, the disadvantage of this method is possible aggregated formation during sample preparation. Initially, formvar-coated grids were floated on a droplet of the saturated preparation on parafilm, to permit the adsorption of the nanoparticles onto the grid. After blotting the grid with a filter paper, the grid was transferred onto a drop of the negative stain. Following this, the grid was blotted with a filter paper and air dried. Aqueous uranyl acetate solution (2%) was used as a negative stain in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
these experiments. The process of preparation was done under constant vacuum. Finally, the samples were examined in a Model JEM2100 transmission electron microscope (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION The physicochemical properties of the drugs and cyclodextrins tested are shown in Table 2. All four drugs tested are somewhat lipophilic and waterinsoluble with log Po/w ranging between 0.8 and 1.6 and solubility from about 1 mg/mL to 5 mg/mL at pH 7.4, whereas the cyclodextrins are much more hydrophilic. The phase-solubility diagrams of the drugs were determined in the aqueous eye drop medium, an aqueous solution containing 0–20% (w/v) cyclodextrin, 0.02% (w/v) benzalkonium chloride, 0.10% (w/v) EDTA and sufficient sodium chloride to make the solution isotonic. All the phase-solubility diagrams were of type A except the diagrams of hydrocortisone in gCD and 80/20 gCD/HPgCD solutions were of BS-type according to the Higuchi-Connor classification system.22 The slopes of the initial linear sections of the diagrams were determined and both the apparent stability constants (K1:1) and the complexation efficiency (CE) determined from the slope (Tab. 3). In case of amphotericin B the values of K1:1 and K1:2 were determined by nonlinear fitting to the AP profile.23 The slopes of the diclofenac profiles were greater than unity indicating that every drug/cyclodextrin inclusion complex contains one cyclodextrin molecule and more than one diclofenac molecule or more likely, that mixtures of inclusion and noninclusion complexes are being formed.8,15,26 Mixtures of gCD and HPgCD have synergistic solubilizing effects on hydrocortisone and diclofenac (Tab. 3).20 Both indomethacin and amphotericin B have very low affinity for gCD and HPgCD as observed by their very low CE value, indicating that only about 2–7% of the cyclodextrin molecules in the solution are forming complexes with these drug molecules.21 The A-type phase-solubility diagrams with slopes less than unity and the observed stability constants indicate that in most cases one drug molecule forms a complex with one cyclodextrin molecule and that the molecular weights (MWs) of the formed complexes are much less than 3500 Da. Even if a 2:1 diclofenac/ HPgCD complex could be formed its MW would only be 2168 Da. The only exception is the 1:2 DOI 10.1002/jps
1576 200 (dec.) — <10 >500
O
OH
O O
OH
HO
HO
O
HOOH
O
OH HO
HO
H2 N
924.1 > 170 (dec.) 5.5; 10 0.8 0.001 Cl
296.2 357.8 157 158 4.2 4.5 0.8 (at pH 7.4) 1.0 (at pH 7.4) 5.2 (at pH 7.4) 0.8 (at pH 7.2)
O
Cl
362.5 214 (dec.) — 1.6 0.4
H H
H
O
HO
Hydroxypropylated gCD is a mixture of structurally related compounds. The chemical structure shown is only a representative structure. Dec. ¼ decomposition upon heating. The logarithm of the octanol/water partition coefficient.
OH
O O HO
O
N O
H N OH
Molecular weight Melting point (8C)b pKa log Po/wc S0 (mg/mL) in water (at RT)
c
OH
O HO O
HO
O HO
HO
O HO
HO COO H H O OH OH OH OH O H O
O
OH
Cl COOH O HO
Chemical structure
b
O
HO OH O
O
OH
O
OH O
HO OH OH OH
Amphotericin B Indomethacin Diclofenac Hydrocortisone
Table 2. Physicochemical Properties of the Sample Compounds23,32–36
Physicochemical Properties
DOI 10.1002/jps
a
OH
HO
HO
O OH O OH
OH O OH
gCD
1297.1 200 (dec.) — 12 249
O
O
O
O
OH
OH
OH HO
HO
O
O
OH
OH O O
O
O
HO
OH
OH
O
O
HO O
O
OH
O
OH
O
O OH
OH
OH
O
HPgCDa
O
O
OH
OH
OH
OH
CYCLODEXTRINS AS SOLUBILIZERS: FORMATION OF COMPLEX AGGREGATES
723
amphotericin B/gCD and 1:2 amphotericin B/ HPgCD complexes with MW of 3518 and 4076 Da, respectively. Thus, the complexes should be able to permeate a semi-permeable cellophane membrane with molecular weight cutoff (MWCO) of 3500 Da. It should also be remembered that in Atype phase solubility diagrams, where aqueous cyclodextrin solutions are saturated with poorly soluble drug, the concentration of unbound drug is constant and equal to the intrinsic solubility of the drug in the aqueous complexation media. The observed increase in drug solubility is due to formation of water-soluble drug/cyclodextrin complexes. Consequently hydrocortisone (MW 362.5) permeation from aqueous hydrocortisone/HPbCD solution through a cellophane membrane with MWCO of 500 is constant and independent of the total hydrocortisone solubility while the permeability through comparable membranes with MWCO of 6–8000 and 12–14,000 increase with increasing solubility up to HPbCD concentration of about 10% before leveling off.8,16 Figure 1 shows the phase-solubility diagrams of the drugs in the aqueous eye drop medium and their permeability profiles from the eye drop medium through MWCO 3500 semi-permeable cellophane membrane. According to Fick’s first law, the flux (J) should be proportional to the concentration of dissolved drug (Cd) in the complex medium (the eye drop medium): J ¼ PCd
(7)
where P is the permeability coefficient. Linear phase-solubility profile indicates that the concentration of dissolved drug in the eye drop medium is proportional to the cyclodextrin concentration (Fig. 1). If the MW of the water-soluble drug/ cyclodextrin complex formed is less than 3500 Da then a plot of J versus Cd should in theory be linear. In other words if the phase-solubility is linear and if the MW of the complex is less than 3500 Da then a linear relationship between the cyclodextrin concentration and J through a MWCO 3500 membrane should be observed. However, all the permeability profiles in Figure 1 show negative deviation from linearity. The profiles are linear up to cyclodextrin concentration of about 5–10% (w/v). As the cyclodextrin concentration increases the profiles start to level off and eventually J becomes independent of the amount of drug dissolved in the aqueous eye drop medium for drugs that display type A phasesolubility profiles, whereas J decreases for drugs displaying BS-type profiles. For the water-soluble JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Table 3. Apparent Stability Constant (K1:1, K1:2), and the Complexation Efficiency (CE) of Selected Drug/Cyclodextrin Complexes in the Aqueous Eye Drop Medium at Room Temperature (22–238C) Cyclodextrin Hydrocortisone gCD gCD/HPgCD gCD/HPgCD HPgCD Diclofenac sodium gCD gCD/HPgCD gCD/HPgCD HPgCD Indomethacin gCD gCD/HPgCD gCD/HPgCD HPgCD Amphotericin B gCD gCD/HPgCD gCD/HPgCD HPgCD
Ratio
Slopea
Correlation Coefficient
K1:1 (M1)
K1:2 (M1)
CE
— 80/20 20/80 —
0.238 0.656 0.617 0.547
0.999 0.994 0.996 0.998
240 1460 1240 925
— — — —
0.31 1.90 1.61 1.21
— 80/20 20/80 —
1.560 1.664 1.579 1.611
0.999 0.998 0.997 0.998
—b —b —b —b
— — — —
—b —b —b —b
— 80/20 20/80 —
0.028 0.024 0.031 0.035
0.944 0.966 0.972 0.944
25 21 28 31
— — — —
0.03 0.02 0.03 0.04
— 80/20 20/80 —
0.063 0.063 0.048 0.040
0.982 0.973 0.960 0.912
1330 1180 1090 1060
150 170 140 120
0.07 0.07 0.05 0.04
The intrinsic solubility (S0) of hydrocortisone, diclofenac sodium, indomethacin and amphotericin B in the eye drop medium was determined to be 0.47 mg/mL (1.3 mM), 6.18 mg/mL (19.4 mM), 0.41 mg/mL (1.2 mM), and 0.29 mg/mL (0.3 mM), respectively, at room temperature. a For indomethacin this is the initial linear slope of the phase-solubility diagram. Hydrocortisone formed BS-type phase-solubility diagrams with gCD and 80/20 gCD/HPgCD and thus the slope was calculated from the initial linear section of the profile. b Could not calculate. The slope of the phase-solubility diagram was greater than unity indicating that the complex contained more than one drug molecule for every cyclodextrin molecule.
drug/cyclodextrin complexes (those that have type A profiles) virtually all increase in solubility at or above 10% (w/v) cyclodextrin concentration is due to formation of complex aggregates with MW greater than about 3500 Da. From the phasesolubility diagrams it can be estimated, based on the hydrocortisone flux through the membrane, that in aqueous HPgCD and gCD/HPgCD solutions saturated with hydrocortisone about 50% the hydrocortisone/cyclodextrin complexes are forming aggregates at 20% cyclodextrin concentration, about 20% at 10% cyclodextrin concentration and about 10% at 5% concentration. Thus, complex aggregation appears to increase with increasing cyclodextrin concentration. The flux of hydrocortisone and indomethacin through semipermeable cellophane membranes with different MWCO is shown in Figure 2. It can be seen that the flux profiles of the hydrocortisone/gCD complex is in agreement with the BS-type profiles (Fig. 1A) that level off at gCD concentration of about 2.5% (w/v). The 80/20 gCD/HPgCD mixture forms somewhat more water-soluble complexes JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
and gives profiles that level off when the cyclodextrin concentration approaches 5% (w/v). The drug solubility decreases at cyclodextrin concentration of about 10% (w/v) which is again reflected in the permeability profiles. Indomethacin forms water-soluble complexes with both pure gCD and the gCD/HPgCD mixture, up to cyclodextrin concentrations 15–20% (w/v), as can be seen from the A-type phase-solubility diagrams (Fig. 1C). Still the increase in J through the MWCO 3500 membrane is insignificant and relatively sharp break is observed in the linear J versus % cyclodextrin profile through the MWCO 6–8000 and 12–14,000 membranes at 5% (w/v) cyclodextrin concentration (Fig. 2). This indicates that although clear solutions are obtained after filtration through 0.45 mm membrane filter, and although A-type phase-solubility profiles are obtained, the water-soluble drug/cyclodextrin complexes formed are unable to permeate membranes with a MWCO of up to 14,000 Da. For hydrocortisone and indomethacin an aggregate with apparent MW of 14,000 Da will contain seven DOI 10.1002/jps
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Figure 2. The effect of cyclodextrin concentration on the flux of hydrocortisone (A) and indomethacin (B) from the aqueous eye drop medium through semipermeable cellophane membrane: MWCO 3500 (&); MWCO 6-8000 (*); and MWCO 12–14,000 (&). The cyclodextrin-containing aqueous eye drop medium was saturated with the drug.
Figure 1. Phase solubility profiles in the aqueous eye drop media and the flux (J) profiles from the eye drop media through MWCO 3500 semipermeable cellophane membrane at room temperature. The cyclodextrincontaining eye drop media was saturated with the drug: hydrocortisone (A); diclofenac sodium (B); indomethacin (C); and amphotericin B (D). gCD (&); 80/20 gCD/HPgCD (&); 20/80 gCD/HPgCD (*); and HPgCD (*).
to eight 1:1 drug/cyclodextrin complexes. Furthermore, all the profiles in Figure 2 show a relative sharp deviation from linearity at cyclodextrin concentration of about 5% (w/v) which indicates that complex monomers are in direct equilibrium with aggregates containing not less than seven to eight drug/cyclodextrin complexes and in similar fashion as when individual surfactant molecules are in equilibrium will micelles. In other words, DOI 10.1002/jps
that contribution of drug/cyclodextrin dimers to heptamers to the observed flux is negligible although such aggregates are able to permeate the MWCO 12–14,000 membrane. The osmolalities of aqueous cyclodextrin solutions were determined at room temperature ( 238C). The solutions contained 0–20% (w/v) gCD, HPgCD or gCD/HPgCD mixtures (80/20 and 20/80 weight mixtures), or up to the saturation region in the case of gCD and gCD/HPgCD (80/20). One experimental set consisted of pure aqueous cyclodextrin solutions (Fig. 3A), while the other set contained 0.2 mg/mL hydrocortisone in identical cyclodextrin solutions (Fig. 3B). Some literature data describing osmotic properties of the studied cyclodextrins are available. There is a principal difference between our results and data obtained by Miyajima et al.,27 which observed a decrease in the value of osmotic and activity coefficients with increasing gCD concentration. Our results are similar to those of Terdale et al.28 in case of aCD. However, an algorithm of osmotic coefficients calculation from vapor pressure measurements was not described which makes discussion of possible reasons of such disagreement impossible. In addition, the osmotic studies of HPgCD in a wide concentration range have been carried out by Zannou et al.29 Their JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Figure 3. The experimental osmotic coefficients versus cyclodextrin molality at room temperature ( 238C). Cyclodextrin solutions without hydrocortisone (A); cyclodextrin solutions containing 0.2 mg/mL of hydrocortisone (B). gCD (^); gCD/HPgCD 80/20 (D); gCD/HPgCD 20/80 (); HPgCD (&).
experimental osmolality values for 0–20% (w/v) HPgCD lie between 0 and 200 mOsm/kg that is in agreement with our observations. The activity coefficients were estimated using Eq. (6) and data from Table 4. The concentration dependences of activity coefficients for all studied solutions are shown in Figure 4. The solute activity coefficients increase with concentration showing nonideal behavior of cyclodextrins in the
studied systems (Fig. 4A) which is in agreement with published data for aggregation of aCD.28 The solutions can be arranged according to increasing deviation from ideality: gCD < 80/20 gCD/HPgCD < 20/80 gCD/HPgCD < HPgCD. Such deviations of activity coefficients from unity can be due to either ‘‘solute–solvent’’ interaction, that is, hydration, or ‘‘solute–solute’’ interaction, that is, association or aggregation, or both. The deviations from ideality are here mainly related to the hydration process (namely, to hydrogen-bond formation between oxygen atoms of the solute and water, and hydrogen-bond formation in the peripheral hydration) but the deviation is also related, although to lesser degree, to solute– solute interactions.28,30 The hydration results in increase in the activity coefficients but the aggregation in a decrease. The observed increase in the activity coefficients with increasing solute concentration can be explained as follows. In the aqueous cyclodextrin solutions a competition between hydration and aggregation processes is taking place where hydration prevails over aggregation leading to increase in activity coefficients above unity. Less positive deviation of activity coefficient is observed for pure gCD (Fig. 4A) and, in the context of competition, it means that the ratio between hydration and aggregation terms is shifted in favor of the aggregation in comparison to the other systems. The positive deviation increases with increasing gCD and HPgCD concentrations and the deviation is greater for HPgCD than for the parent gCD. This is because HPgCD is a somewhat larger molecule than gCD which allows for larger number of hydrogen-bonds (i.e., greater hydration) and consequently hydration/aggregation ratio is shifted in favor of the hydration. Regarding solutions containing cyclodextrins and hydrocortisone the solutions can be arranged according
Table 4. Parameters of Eq. (5) for the Studied Aqueous Solutions at Room Temperature ( 238C) Solution gCD 80/20 gCD/HPgCD 20/80 gCD/HPgCD HPgCD gCD þ HC 80/20 gCD/HPgCD þ HC 20/80 gCD/HPgCD þ HC HPgCD þ HC
B1
B2
B3
R
s 102
n
8 1 10 2 15 2 18 1
077 19 104 27 172 33 214 18
270 080 383 121 759 167 995 095
0.9913 0.9902 0.9948 0.9991
1.89 2.55 2.49 1.27
7 7 7 7
7 1 12 4 10 3 11 2
075 27 587 225 140 60 111 39
— 6200 3300 600 300 370 200
0.9858 0.9422 0.9363 0.9878
1.00 1.98 4.43 2.70
6 7 6 6
R, pair correlation coefficient; s, standard deviation; n, number of points. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
DOI 10.1002/jps
CYCLODEXTRINS AS SOLUBILIZERS: FORMATION OF COMPLEX AGGREGATES
Figure 4. The estimated activity coefficients of solute versus molality of the cyclodextrins studied in aqueous solutions at room temperature ( 238C). Cyclodextrins without hydrocortisone (A); cyclodextrins with 0.2 mg/mL of hydrocortisone (B). gCD (—); gCD/HPgCD 80/20 (- - -); gCD/HPgCD 20/80 (- - -); HPgCD (. . .).
to increasing deviation from ideality: 80/20 gCD/ HPgCD < gCD < 20/80 gCD/HPgCD < HPgCD. Figure 4B shows that the activity coefficients of all tested drug containing cyclodextrin solutions have in general smaller values than comparable solutions without the drug (Fig. 4A). This is due to the fact that in addition to hydration and aggregation the complexation process takes place in these drug containing solution systems. The complexation causes additional decrease in the value of the activity coefficients. This conclusion is in agreement with complexation data from Table 3 where the 80/20 gCD/HPgCD mixture has the largest CE as well as the largest negative deviation of activity coefficients (Fig. 4B). It would be interesting to determine the driving forces of aggregation process via more detailed thermodynamic analysis that is by estimation of excess thermodynamic parameters of solutes (the Gibbs energy, enthalpy and entropy). This is a matter of our future studies. Transmission electron microscopic (TEM) micrographs of 10% (w/v) gCD and HPgCD solutions, as well as of aqueous 10% (w/v) 80/20 DOI 10.1002/jps
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and 20/80 mixtures of gCD and HPgCD, saturated with hydrocortisone are shown in Figure 5. The sizes of hydrocortisone/gCD and hydrocortisone/ HPgCD complex aggregates are approximately 10–50 and 10–80 nm, respectively (Fig. 5A and B). The aggregate diameter of the hydrocortisone/ (gCD/HPgCD) complex mixtures is much larger than that of gCD and HPgCD or up to couple of hundred nm in diameter, and appeared to be formed by aggregation of smaller complex aggregates (Fig. 5C and D). Previously we have shown that addition of relatively small amounts of the water-soluble HPgCD to aqueous gCD formulations increases the CE of gCD and results in synergistic effect with regard to drug solubilization in aqueous media.20 It is possible that these effects are related to formation of larger aggregates. Other investigators have shown by TEM imaging that the natural a-cyclodextrin (aCD), b-cyclodextrin (bCD) and gCD, and their complexes, form aggregates in pure aqueous solutions.12–14,31 However, these were dilute cyclodextrin solutions (1%, w/v) and the weight fractions of the aggregates were only between 0.0002% and 0.002% of the total cyclodextrin dissolved in the solution.13 Our permeation studies indicate that the weight fraction of the aggregates gradually increases with increasing cyclodextrin concentration, especially when the cyclodextrin concentration exceeds 5% (Figs. 1 and 2). We have made similar observations in aqueous HPbCD solutions.8,15,16
CONCLUSIONS The natural aCD, bCD and gCD, their derivatives, such as HPbCD and HPgCD, and complexes self-assemble to form nanoscale aggregates in aqueous solutions. At cyclodextrin concentration of about 1% (w/v) or lower the relative mass contribution of these aggregates is less than 0.01% but it gradually increases with increasing cyclodextrin concentration until at about 5–10% (w/v) when all increase in dissolved drug/cyclodextrin complexes is in the form of cyclodextrin aggregates. In most cases these solutions do not display static light scattering and appear to the naked eye to be clear solutions and, thus, the diameter of the aggregates is below couple of hundred nanometers. Our studies indicate that most frequently the size is between 10 and 100 nm which is in an agreement with previous studies by other investigators.13,14 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Figure 5. Transmission electron microscopic images of saturated solution of hydrocortisone in 10% cyclodextrin solutions: aggregated hydrocortisone/gCD complexes (A); hydrocortisone/HPgCD complexes (B); hydrocortisone/(gCD/HPgCD) complexes with gCD/HPgCD ratio of 80/20 (C); and hydrocortisone/(gCD/HPgCD) complexes with gCD/HPgCD ratio of 20/80 (D).
ACKNOWLEDGMENTS Financial support from the Eimskip fund, Iceland, is gratefully acknowledged. We gratefully acknowledge the support of Scientific and Technology Research Equipment Centre, Chulalongkorn University, Bangkok, Thailand for TEM analysis.
REFERENCES 1. Loftsson T, Ducheˆ ne D. 2007. Cyclodextrins and their pharmaceutical applications. Int J Pharm 329:1–11. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
2. Dodziuk H, editor. 2006. Cyclodextrins and their complexes. 1st edition. Weinheim: Wiley-VCH Verlag. 3. Kajta´ r M, Vikmon M, Morlin E, Szejtli J. 1989. Aggregation of amphotericin B in the presence of g-cyclodextrin. Biopolymers 28:1585–1596. 4. Coleman AW, Nicolis I, Keller N, Dalbiez JP. 1992. Aggregation of cyclodextrins: An explanation of the abnormal solubility of b-cyclodextrin. J Incl Phenom Macroc Chem 13:139–143. 5. Andronati SA, Shapiro YE, Yakubovskaya LN, Gorbatyuk VY, Andronati KS, Krasnoschekaya SP. 1996. Inclusion compounds of psychotropic agents and cyclodextrins. J Incl Phenom Macroc Chem 24:175–186. 6. Gabelica V, Galic N, De Pauw E. 2002. On the specificity of cyclodextrin complexes detected by DOI 10.1002/jps
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electrospray mass spectrometry. J Am Soc Mass Spectrom 13:946–953. Correia I, Bezzenine N, Ronzani N, Platzer N, Beloeil J-C, Doan B-T. 2002. Study of inclusion complexes of acridine with b- and (2,6-di-O-methyl)-b-cyclodextrin by use of solubility diagrams and NMR spectroscopy. J Phys Org Chem 15:647–659. Loftsson T, Ma´ sson M, Brewster ME. 2004. Selfassociation of cyclodextrins and cyclodextrin complexes. J Pharm Sci 93:1091–1099. Bika´ di Z, Kurdi R, Balogh S, Szema´ n J, Hazai E. 2006. Aggregation of cyclodextrins as an important factor to determine their complexation behavior. Chem Biodiv 3:1266–1276. Yan P, Tang J-N, Xiao J-X. 2007. Interaction between b-cyclodextrin and mixed cationic-anionic surfactants (2): Aggregation behavior. J Dispers Sci Technol 28:623–626. Szente L, Szejtli J, Kis GL. 1998. Spontaneous opalescence of aqueous g-cyclodextrin solutions: Complex formation or self-aggregation. J Pharm Sci 87:778–781. Wu A, Shen X, He Y. 2006. Investigation of gcyclodextrin nanotube induced by N,N’-diphenylbenzidine molecule. J Coll Interf Sci 297:525–533. Wu A, Shen X, He Y. 2006. Micrometer-sized rodlike structure formed by the secondary assembly of cyclodextrin nanotube. J Coll Interf Sci 302:87–94. Bonini M, Rossi S, Karlsson G, Almgren M, Lo Nostro P, Baglioni P. 2006. Self-assembly of b-cyclodextrin in water. Part 1. Cryo-TEM and dynamic and static light scattering. Langmuir 22: 1478–1484. Loftsson T, Magnu´ sdo´ ttir A, Ma´ sson M, Sigurjo´ nsdo´ ttir JF. 2002. Self-association and cyclodextrin solubilization of drugs. J Pharm Sci 91:2307–2316. Loftsson T, Ma´ sson M, Sigurdsson HH. 2002. Cyclodextrins and drug permeability through semipermeable cellophane membranes. Int J Pharm 232:35–43. Loftsson T, Ma´sson M. 2004. The effects of watersoluble polymers on cyclodextrins and cyclodextrin solubilization of drugs. J Drug Del Sci Tech 14:35–43. Redenti E, Szente L, Szejtli J. 2000. Drug/cyclodextrin/hydroxy acid multicomponent systems. Properties and pharmaceutical applications. J Pharm Sci 89:1–8. Yamakawa T, Nishimura S. 2003. Liquid formulation of a novel non-fluorinated topical quinolone, T3912, utilizing the synergic solubilizing effect of the combined use of magnesium ions and hydroxypropyl-b-cyclodextrin. J Control Rel 86:101–113. Jansook P, Loftsson T. 2008. gCD/HPgCD: Synergistic solubilization. Int J Pharm 363:217–219. Loftsson T, Hreinsdo´ ttir D, Ma´ sson M. 2005. Evaluation of cyclodextrin solubilization of drugs. Int J Pharm 302:18–28. Higuchi T, Connors KA. 1965. Phase-solubility techniques. Adv Anal Chem Instrum 4:117–212.
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23. Brewster ME, Loftsson T. 2007. Cyclodextrins as pharmaceutical solubilizers. Adv Drug Deliv Rev 59:645–666. 24. Saarinen-Savolainen P, Urtti A, Jarho P, Ja¨ rvinen T. 1997. b-Cyclodextrin derivatives (2-HP-b-CD, SBE4-b-CD) decrease the amphiphilicity and membrane perturbing effects of pilocarpine prodrugs. Eur J Pharm Sci 5:89–96. 25. Bugler J, Sommerdijk NAJM, Visser AJWG, van Hoek A, Nolte RJM, Engbersen JFJ, Reinhoudt DN. 1999. Interconnective host-guest complexation of beta-cyclodextrin-calix[4]arene couples. J Am Chem Soc 121:28–33. 26. Magnusdottir A, Ma´ sson M, Loftsson T. 2002. Self association and cyclodextrin solubilization of NSAIDs. J Incl Phenom Macroc Chem 44:213–218. 27. Miyajima K, Sawada M, Nakagaki M. 1983. Viscosity B-coefficients, apparent molar volumes, and activity coefficients for a- and g-cyclodextrins in aqueous solutions. Bull Chem Soc Jpn 56:3556– 3560. 28. Terdale SS, Dagade DH, Patil KJ. 2006. Thermodynamic studies of molecular interactions in aqueous alpha-cyclodextrin solutions: Application of McMillan-Mayer and Kirkwood-Buff theories. J Phys Chem B 106:18583–18593. 29. Zannou EA, Streng WH, Stella VJ. 2001. Osmotic properties of sulfobutylether and hydroxypropyl cyclodextrins. Pharm Res 18:1226–1231. 30. Patil K, Pawar R, Dagade D. 2002. Studies of osmotic and activity coefficients in aqueous and CCl4 solutions of 18-Crown-6 at 258C. J Phys Chem A 106:9606–9611. 31. He W, Fu P, Shen XH, Gao HC. 2008. Cyclodextrinbased aggregates and characterization by microscopy. Micron 39:495–516. 32. Moffat AC, Osselton MD, Widdop B, editors. 2004. Clarke’s Analysis of drugs and poisons. 3rd edition. London: Pharmaceutical Press. 33. Lavasanifar A, Samuel J, Kwon GS. 2002. The effect of fatty acid substitution on the in vitro release of amphotericin B from micelles composed of poly(ethylene oxide)-block-poly(N-hexyl stearate-L-aspartamide). J Control Rel 79:165–172. 34. Nokhodchi A, Javadzadeh Y, Siahi-Shadbad MR, Barzegar-Jalali M. 2005. The effect of type and concentration of vehicles on the dissolution rate of a poorly soluble drug (indomethacin) from liquisolid compacts. J Pharm Pharmaceut Sci 8:18–25. 35. Riess W, Stierlin H, Degen P, Faigle JW, Gerardin A, Moppert J, Sallmann A, Schmid K, Schweizer A, Sulc M, Theobald W, Wagner J. 1978. Pharmacokinetics and metabolism of the anti-inflammatory agent Voltaren. Scand J Rheumato Suppl 22:17–29. 36. Huang Y-B, Wu P-C, Ko H-M, Tsai Y-H. 1995. Cardamom oil as a skin permeation enhancer for indomethacin, piroxicam and diclofenac. Int J Pharm 126:111–117.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
Received 5 March 2009; revised 19 May 2009; accepted 20 May 2009 Published online 7 August 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21861
ABSTRACT: Three different techniques were applied to investigate aggregation of drug/ cyclodextrin complexes, that is, drug permeation through semi-permeable membranes, determination of changes in the value of activity coefficients of drug/cyclodextrin complex solutions and transmission electron microscopy (TEM). The aqueous solutions studied contained g-cyclodextrin, 2-hydroxypropyl-g-cyclodextrin or mixtures thereof, and hydrocortisone, amphotericin B, diclofenac sodium or indomethacin. The permeation studies indicated that drug/cyclodextrin complex monomers (i.e., unaggregated complexes) were dominating at cyclodextrin concentrations below 5% (w/v). Then formation of aggregates gradually increased with increasing cyclodextrin concentration until all increase in dissolved drug/cyclodextrin complexes was due to formation of cyclodextrin aggregates. This happened even though the observed phase-solubility diagrams were linear, that is, were of AL-type. The activity coefficients showed positive deviation from ideal state. This positive deviation is due to concurrent of several processes, that is, hydration, aggregation and complex formation. The observed deviations from ideality indicated that complex aggregates were formed in the aqueous complexation media. TEM pictures showed formation of aggregates in both pure aqueous cyclodextrin solutions as well as in cyclodextrin solutions that had been saturated with hydrocortisone. The aggregate diameter was between 10 and 100 nm but larger aggregates with diameter of about 200 nm were formed through assemble of smaller aggregates. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:719–729, 2010
Keywords:
cyclodextrin; complex; aggregate; permeation; activity coefficient; TEM
INTRODUCTION Cyclodextrins are hydrophilic cyclic oligosaccharides with a lipophilic central cavity. In aqueous solutions cyclodextrins are able to solubilize many hydrophobic drugs by taking up some lipophilic moiety of the drugs into the cavity, that is, through formation of water-soluble inclusion complexes. Aqueous cyclodextrin solutions are frequently used as nontoxic solvents during screening of new chemical entities (NCE) both Correspondence to: Thorsteinn Loftsson (Telephone: þ354525-4464; Fax: þ354-525-4071; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 719–729 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
in vitro and in vivo, and cyclodextrins can be found in just over 30 marketed drug products worldwide.1 In aqueous solutions free drug and cyclodextrin molecules are in dynamic equilibrium with the complexes and every complex unity is generally assumed to be free and independent of other complexes as well as of other excipients found in the solution.2 It is however becoming increasingly apparent that such assumptions may not be universally applicable or all encompassing. Specifically, there is a growing body of evidence that supports the important contribution of noninclusion aspects for drug solubilization by cyclodextrins including surfactant-like effects and molecular aggregation, that is, formation of particulate systems.3–10 It is
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known that spontaneous opalescence occurs in aqueous g-cyclodextrin (gCD) solutions during storage11 and investigations of aqueous 1% (w/v) gCD solutions have shown that about 0.02% (w/w) of gCD in the solutions is in the form of aggregates.12 Comparable observations have been made in a-cyclodextrin (aCD) and b-cyclodextrin (bCD) solutions.13,14 The diameter of these cyclodextrin aggregates is approximately 100 nm. The cyclodextrin derivatives, such as 2-hydroxypropyl-b-cyclodextrin (HPbCD), are also thought to form aggregates in aqueous solutions.8 In aqueous solutions the relative amount of aggregated cyclodextrin increases with increasing cyclodextrin concentration and permeation studies of hydrocortisone/HPbCD solutions have indicated that in spite of the transparent nature of the solutions virtually all increase in drug solubility observed at HPbCD concentrations above 10% (w/v) is due to hydrocortisone/HPbCD aggregate formation.8,15,16 However, aggregates of the natural aCD, bCD, and gCD were studied in very dilute (about 1%, w/ v) aqueous cyclodextrin solutions and the hydrocortisone/HPbCD aggregate formation was only observed by permeation studies. In this present study three different techniques are used to verify formation of the aggregates. Water-soluble polymers, metal ions and various carboxylic acids are also known to influence the cyclodextrin solubilization of drugs, possibly through interactions with cyclodextrin aggregates.17–19 Previously we have shown that addition of relatively small amounts of the water-soluble 2-hydroxypropyl-g-cyclodextrin (HPgCD) to aqueous gCD formulations increases the complexation efficiency (CE) of gCD and reduces the turbidity of gCD solutions. Mixing HPgCD with gCD resulted in synergistic effect with regard to drug solubilization in aqueous media.20 It was hypothesized that the observed effect was somehow related to formation of gCD and HPgCD nanoparticles. The purpose of this present study was to investigate further the solubilizing effects of gCD, HPgCD, and gCD/HPgCD mixtures and their formations of nanosize aggregates.
MATERIALS AND METHODS Materials Hydrocortisone (HC) was purchased from ICN Biomedicals (Aurora, OH), amphotericin B (AmB), JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
diclofenac sodium (DC-Na) and indomethacin (IDM) from Sigma (St. Louis, MO), g-cyclodextrin (gCD) and 2-hydroxypropyl-g-cyclodextrin (HPgCD) MS 0.6 (MW 1576 Da) from Wacker Chemie (Munich, Germany), disodium edetate dehydrate (EDTA) and sodium chloride from Merck (Darmstadt, Germany), and benzalkonium chloride from Sigma. Semi-permeable cellophane membranes (SpectaPor1, molecular weight cut-off (MWCO) 3500, 6–8000, 12–14,000) was purchased from Spectrum Europe (Breda, Netherlands). All other chemicals used were of analytical reagent grade purity. Milli-Q (Millipore, Billerica, MA) water was used for the preparation of all solutions.
Phase-Solubility Profiles The solubility of the drugs was determined by previously described heating method.21 Hydrocortisone, diclofenac sodium and indomethacin were chemically stable (i.e., their degradation was less than 1%) during heating (1218C for 20 min) in aqueous gCD and HPgCD solutions. However amphotericin B was chemically unstable during heating in an autoclave and thus amphotericin B solutions were saturated through heating in a sonicator to 608C for 30 min. The suspension was heated to promote drug, and in some case cyclodextrin, saturation of the eye drop complexation medium. Excess amount of the drug to be tested was added to a solution containing 0–20% (w/v) cyclodextrin in eye drop vehicle consisting of aqueous solution containing 0–20% (w/v) cyclodextrin, benzalkonium chloride (0.02%, w/v), EDTA (0.1%, w/v) and sufficient sodium chloride to obtain isotonicity. The drug suspensions were heated in an autoclave (1218C, 20 min), or in the case of amphotericin B to 608C for 30 min in a sonicator, and allowed to cool to room temperature. Then a small amount of solid drug was added to the suspensions to promote drug precipitation and the pH adjusted to 7.4 with concentrated sodium hydroxide solution. The pH was periodically tested during the equilibration time and the samples were ready to be analyzed. The suspensions were equilibrated at room temperature (22–238C) for 7 days under constant agitation. Preliminary experiments showed that 7 days are more than enough time to reach solubility equilibrium. After equilibrium was attained, the suspensions were filtered through 0.45 mm cellulose membrane filter, the filtrates diluted with the mobile phase and analyzed by HPLC. The DOI 10.1002/jps
CYCLODEXTRINS AS SOLUBILIZERS: FORMATION OF COMPLEX AGGREGATES
apparent complexation constants for drug/cyclodextrin complexes (K1:1) were determined according to the phase-solubility method of Higuchi and Connors.22 The higher order complexation constants were determined through nonlinear fitting of the AP-type phase-solubility diagrams.22–24 The complexation efficiency (CE) was determined from the slope of linear phase-solubility diagrams (plots of the total drug solubility ([drug]t) versus total cyclodextrin concentration ([CD]t) in moles per liter):21 CE ¼
Slope ½drug=CD complex ¼ 1 Slope ½CD
¼ K1:1 S0
(1)
where K1:1 is the stability constant of the drug/ cyclodextrin 1:1 complex and S0 is the intrinsic solubility of the drug.
Quantitative Determinations The quantitative determinations of the individual drugs were performed on a reversed-phase HPLC component system from Hewlett Packard Series 1100, consisting of a G132A binary pump with a G1379A solvent degasser, a G13658 multiple wavelength detector, a G1313A auto sampler, and Phenomenex Luna 5 mm C18 reverse-phase column (150 mm 4.6 mm). The HPLC chromatographic conditions were shown in Table 1.
721
membrane. Before usage the membrane was soaked overnight in the receptor phase that consisted of pH 7.4 phosphate buffer saline containing 5% (w/v) mixture of gCD/HPgCD (50/ 50 weight mixtures). To avoid possibility of membrane fouling the receptor phase was autoclaved at 1218C for 20 min prior to use. Cyclodextrin was added to the receptor phase to ensure sufficient drug solubility to maintain sink condition throughout the experiment. The receptor phase was sonicated under vacuum to remove dissolved air before it was placed in the receptor chamber. The donor phase (2 mL) consisted of 0–20% (w/v) cyclodextrin solution in eye drop vehicle saturated with the drug, as previously described under methods in the phase-solubility section. The study was conducted at room temperature (22–238C) and under continuous stirring for 6 h by a magnetic stirring bar rotating at 300 rpm. An aliquot of receptor medium (150 mL) was withdrawn at 30, 60, 120, 180, 240, and 360 min and replaced immediately with an equal volume of fresh receptor phase. The drug concentration in the receptor phase was determined by HPLC. The steady state flux was calculated as the slope (dq/dt) of linear section of the amount of drug in the receptor chamber (q) versus time (t) profiles, and the apparent permeability coefficient ( Papp) was calculated from the flux (J) according to Eq. (2): J¼
dq ¼ Papp Cd A dt
(2)
where A is the surface area of the mounted membrane (1.77 cm2) and Cd is the initial concentration of the drug in the donor phase.
Permeation Studies The permeability of individual drugs was carried out using Franz diffusion cell apparatus consisted of a donor and a receptor compartment (FDC 400 15FF, Vangard International, Neptune, NJ). The donor chamber and the receptor compartment were separated by a semi-permeable cellophane
Determination of Activity Coefficients The concentration dependences of osmolalities were measured using Knauer K-7000 vapor pressure osmometer (Knauer, Germany). The
Table 1. HPLC Conditions Drugs Hydrocortisone Diclofenac sodium Indomethacin Amphotericin B
Mobile Phasea ACN:THF:water (33:1:66) ACN:1.0% acetic acid (60:40) ACN:0.5% acetic acid (50:50) ACN:0.25 mM EDTA (37:63)
Flow Rate (mL/min) Wavelength (nm) Retention Time (min) 1.5 1.5 1.5 1.0
241 282 240 403
5.1 4.0 6.9 3.2
ACN, acetonitrile; THF, tetrahydrofuran; acetic acid, aqueous acetic acid solution; EDTA, aqueous disodium edetate dehydrate solution. a Volume ratios. DOI 10.1002/jps
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osmometer was calibrated with standard sodium chloride solutions of known osmolality. The osmotic coefficients, w, were calculated from osmolalities by equation: Osm ¼ n’m
(3)
where Osm is osmolality of a solute in osmol/kg, n is the number of particles in which the solute molecules dissociate (i.e., n ¼ 1 when there is no dissociation, like in our case), and m is the molal concentration of a solute. The activity coefficients, g, of solutes in the solutions studied were estimated using Bjerrum equation: Zm ln g ¼ ’ 1 þ ð’ 1Þ d ln m (4) 0
The concentration dependency of osmotic coefficients is described by appropriate polynomial function, that is: ’ ¼ 1 þ B1 m þ B2 m2 þ B3 m3 þ
(5)
Then, Eq. (5) is introduced into Eq. (4) instead of w, which results in: 3 4 ln g ¼ 2B1 m þ B2 m2 þ B3 m3 þ 2 3
(6)
As follows from Eqs. (5) and (6) the standard state is represented by an ideal solution with m ! 0, for which both w and g ! 1.
Transmission Electron Microscopy (TEM) Analysis The morphology and size of the aggregates in hydrocortisone saturated aqueous 10% (w/v) gCD, HPgCD and gCD/HPgCD (80/20 and 20/80 weight mixtures) solutions were analyzed using a transmission electron microscope. One of the easiest ways of preparing the samples is by negative staining. This preparation method is useful for visualizing suspensions of small particles. The aggregates were visualized by TEM using the uranyl staining method.25 However, the disadvantage of this method is possible aggregated formation during sample preparation. Initially, formvar-coated grids were floated on a droplet of the saturated preparation on parafilm, to permit the adsorption of the nanoparticles onto the grid. After blotting the grid with a filter paper, the grid was transferred onto a drop of the negative stain. Following this, the grid was blotted with a filter paper and air dried. Aqueous uranyl acetate solution (2%) was used as a negative stain in JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
these experiments. The process of preparation was done under constant vacuum. Finally, the samples were examined in a Model JEM2100 transmission electron microscope (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION The physicochemical properties of the drugs and cyclodextrins tested are shown in Table 2. All four drugs tested are somewhat lipophilic and waterinsoluble with log Po/w ranging between 0.8 and 1.6 and solubility from about 1 mg/mL to 5 mg/mL at pH 7.4, whereas the cyclodextrins are much more hydrophilic. The phase-solubility diagrams of the drugs were determined in the aqueous eye drop medium, an aqueous solution containing 0–20% (w/v) cyclodextrin, 0.02% (w/v) benzalkonium chloride, 0.10% (w/v) EDTA and sufficient sodium chloride to make the solution isotonic. All the phase-solubility diagrams were of type A except the diagrams of hydrocortisone in gCD and 80/20 gCD/HPgCD solutions were of BS-type according to the Higuchi-Connor classification system.22 The slopes of the initial linear sections of the diagrams were determined and both the apparent stability constants (K1:1) and the complexation efficiency (CE) determined from the slope (Tab. 3). In case of amphotericin B the values of K1:1 and K1:2 were determined by nonlinear fitting to the AP profile.23 The slopes of the diclofenac profiles were greater than unity indicating that every drug/cyclodextrin inclusion complex contains one cyclodextrin molecule and more than one diclofenac molecule or more likely, that mixtures of inclusion and noninclusion complexes are being formed.8,15,26 Mixtures of gCD and HPgCD have synergistic solubilizing effects on hydrocortisone and diclofenac (Tab. 3).20 Both indomethacin and amphotericin B have very low affinity for gCD and HPgCD as observed by their very low CE value, indicating that only about 2–7% of the cyclodextrin molecules in the solution are forming complexes with these drug molecules.21 The A-type phase-solubility diagrams with slopes less than unity and the observed stability constants indicate that in most cases one drug molecule forms a complex with one cyclodextrin molecule and that the molecular weights (MWs) of the formed complexes are much less than 3500 Da. Even if a 2:1 diclofenac/ HPgCD complex could be formed its MW would only be 2168 Da. The only exception is the 1:2 DOI 10.1002/jps
1576 200 (dec.) — <10 >500
O
OH
O O
OH
HO
HO
O
HOOH
O
OH HO
HO
H2 N
924.1 > 170 (dec.) 5.5; 10 0.8 0.001 Cl
296.2 357.8 157 158 4.2 4.5 0.8 (at pH 7.4) 1.0 (at pH 7.4) 5.2 (at pH 7.4) 0.8 (at pH 7.2)
O
Cl
362.5 214 (dec.) — 1.6 0.4
H H
H
O
HO
Hydroxypropylated gCD is a mixture of structurally related compounds. The chemical structure shown is only a representative structure. Dec. ¼ decomposition upon heating. The logarithm of the octanol/water partition coefficient.
OH
O O HO
O
N O
H N OH
Molecular weight Melting point (8C)b pKa log Po/wc S0 (mg/mL) in water (at RT)
c
OH
O HO O
HO
O HO
HO
O HO
HO COO H H O OH OH OH OH O H O
O
OH
Cl COOH O HO
Chemical structure
b
O
HO OH O
O
OH
O
OH O
HO OH OH OH
Amphotericin B Indomethacin Diclofenac Hydrocortisone
Table 2. Physicochemical Properties of the Sample Compounds23,32–36
Physicochemical Properties
DOI 10.1002/jps
a
OH
HO
HO
O OH O OH
OH O OH
gCD
1297.1 200 (dec.) — 12 249
O
O
O
O
OH
OH
OH HO
HO
O
O
OH
OH O O
O
O
HO
OH
OH
O
O
HO O
O
OH
O
OH
O
O OH
OH
OH
O
HPgCDa
O
O
OH
OH
OH
OH
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amphotericin B/gCD and 1:2 amphotericin B/ HPgCD complexes with MW of 3518 and 4076 Da, respectively. Thus, the complexes should be able to permeate a semi-permeable cellophane membrane with molecular weight cutoff (MWCO) of 3500 Da. It should also be remembered that in Atype phase solubility diagrams, where aqueous cyclodextrin solutions are saturated with poorly soluble drug, the concentration of unbound drug is constant and equal to the intrinsic solubility of the drug in the aqueous complexation media. The observed increase in drug solubility is due to formation of water-soluble drug/cyclodextrin complexes. Consequently hydrocortisone (MW 362.5) permeation from aqueous hydrocortisone/HPbCD solution through a cellophane membrane with MWCO of 500 is constant and independent of the total hydrocortisone solubility while the permeability through comparable membranes with MWCO of 6–8000 and 12–14,000 increase with increasing solubility up to HPbCD concentration of about 10% before leveling off.8,16 Figure 1 shows the phase-solubility diagrams of the drugs in the aqueous eye drop medium and their permeability profiles from the eye drop medium through MWCO 3500 semi-permeable cellophane membrane. According to Fick’s first law, the flux (J) should be proportional to the concentration of dissolved drug (Cd) in the complex medium (the eye drop medium): J ¼ PCd
(7)
where P is the permeability coefficient. Linear phase-solubility profile indicates that the concentration of dissolved drug in the eye drop medium is proportional to the cyclodextrin concentration (Fig. 1). If the MW of the water-soluble drug/ cyclodextrin complex formed is less than 3500 Da then a plot of J versus Cd should in theory be linear. In other words if the phase-solubility is linear and if the MW of the complex is less than 3500 Da then a linear relationship between the cyclodextrin concentration and J through a MWCO 3500 membrane should be observed. However, all the permeability profiles in Figure 1 show negative deviation from linearity. The profiles are linear up to cyclodextrin concentration of about 5–10% (w/v). As the cyclodextrin concentration increases the profiles start to level off and eventually J becomes independent of the amount of drug dissolved in the aqueous eye drop medium for drugs that display type A phasesolubility profiles, whereas J decreases for drugs displaying BS-type profiles. For the water-soluble JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Table 3. Apparent Stability Constant (K1:1, K1:2), and the Complexation Efficiency (CE) of Selected Drug/Cyclodextrin Complexes in the Aqueous Eye Drop Medium at Room Temperature (22–238C) Cyclodextrin Hydrocortisone gCD gCD/HPgCD gCD/HPgCD HPgCD Diclofenac sodium gCD gCD/HPgCD gCD/HPgCD HPgCD Indomethacin gCD gCD/HPgCD gCD/HPgCD HPgCD Amphotericin B gCD gCD/HPgCD gCD/HPgCD HPgCD
Ratio
Slopea
Correlation Coefficient
K1:1 (M1)
K1:2 (M1)
CE
— 80/20 20/80 —
0.238 0.656 0.617 0.547
0.999 0.994 0.996 0.998
240 1460 1240 925
— — — —
0.31 1.90 1.61 1.21
— 80/20 20/80 —
1.560 1.664 1.579 1.611
0.999 0.998 0.997 0.998
—b —b —b —b
— — — —
—b —b —b —b
— 80/20 20/80 —
0.028 0.024 0.031 0.035
0.944 0.966 0.972 0.944
25 21 28 31
— — — —
0.03 0.02 0.03 0.04
— 80/20 20/80 —
0.063 0.063 0.048 0.040
0.982 0.973 0.960 0.912
1330 1180 1090 1060
150 170 140 120
0.07 0.07 0.05 0.04
The intrinsic solubility (S0) of hydrocortisone, diclofenac sodium, indomethacin and amphotericin B in the eye drop medium was determined to be 0.47 mg/mL (1.3 mM), 6.18 mg/mL (19.4 mM), 0.41 mg/mL (1.2 mM), and 0.29 mg/mL (0.3 mM), respectively, at room temperature. a For indomethacin this is the initial linear slope of the phase-solubility diagram. Hydrocortisone formed BS-type phase-solubility diagrams with gCD and 80/20 gCD/HPgCD and thus the slope was calculated from the initial linear section of the profile. b Could not calculate. The slope of the phase-solubility diagram was greater than unity indicating that the complex contained more than one drug molecule for every cyclodextrin molecule.
drug/cyclodextrin complexes (those that have type A profiles) virtually all increase in solubility at or above 10% (w/v) cyclodextrin concentration is due to formation of complex aggregates with MW greater than about 3500 Da. From the phasesolubility diagrams it can be estimated, based on the hydrocortisone flux through the membrane, that in aqueous HPgCD and gCD/HPgCD solutions saturated with hydrocortisone about 50% the hydrocortisone/cyclodextrin complexes are forming aggregates at 20% cyclodextrin concentration, about 20% at 10% cyclodextrin concentration and about 10% at 5% concentration. Thus, complex aggregation appears to increase with increasing cyclodextrin concentration. The flux of hydrocortisone and indomethacin through semipermeable cellophane membranes with different MWCO is shown in Figure 2. It can be seen that the flux profiles of the hydrocortisone/gCD complex is in agreement with the BS-type profiles (Fig. 1A) that level off at gCD concentration of about 2.5% (w/v). The 80/20 gCD/HPgCD mixture forms somewhat more water-soluble complexes JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
and gives profiles that level off when the cyclodextrin concentration approaches 5% (w/v). The drug solubility decreases at cyclodextrin concentration of about 10% (w/v) which is again reflected in the permeability profiles. Indomethacin forms water-soluble complexes with both pure gCD and the gCD/HPgCD mixture, up to cyclodextrin concentrations 15–20% (w/v), as can be seen from the A-type phase-solubility diagrams (Fig. 1C). Still the increase in J through the MWCO 3500 membrane is insignificant and relatively sharp break is observed in the linear J versus % cyclodextrin profile through the MWCO 6–8000 and 12–14,000 membranes at 5% (w/v) cyclodextrin concentration (Fig. 2). This indicates that although clear solutions are obtained after filtration through 0.45 mm membrane filter, and although A-type phase-solubility profiles are obtained, the water-soluble drug/cyclodextrin complexes formed are unable to permeate membranes with a MWCO of up to 14,000 Da. For hydrocortisone and indomethacin an aggregate with apparent MW of 14,000 Da will contain seven DOI 10.1002/jps
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Figure 2. The effect of cyclodextrin concentration on the flux of hydrocortisone (A) and indomethacin (B) from the aqueous eye drop medium through semipermeable cellophane membrane: MWCO 3500 (&); MWCO 6-8000 (*); and MWCO 12–14,000 (&). The cyclodextrin-containing aqueous eye drop medium was saturated with the drug.
Figure 1. Phase solubility profiles in the aqueous eye drop media and the flux (J) profiles from the eye drop media through MWCO 3500 semipermeable cellophane membrane at room temperature. The cyclodextrincontaining eye drop media was saturated with the drug: hydrocortisone (A); diclofenac sodium (B); indomethacin (C); and amphotericin B (D). gCD (&); 80/20 gCD/HPgCD (&); 20/80 gCD/HPgCD (*); and HPgCD (*).
to eight 1:1 drug/cyclodextrin complexes. Furthermore, all the profiles in Figure 2 show a relative sharp deviation from linearity at cyclodextrin concentration of about 5% (w/v) which indicates that complex monomers are in direct equilibrium with aggregates containing not less than seven to eight drug/cyclodextrin complexes and in similar fashion as when individual surfactant molecules are in equilibrium will micelles. In other words, DOI 10.1002/jps
that contribution of drug/cyclodextrin dimers to heptamers to the observed flux is negligible although such aggregates are able to permeate the MWCO 12–14,000 membrane. The osmolalities of aqueous cyclodextrin solutions were determined at room temperature ( 238C). The solutions contained 0–20% (w/v) gCD, HPgCD or gCD/HPgCD mixtures (80/20 and 20/80 weight mixtures), or up to the saturation region in the case of gCD and gCD/HPgCD (80/20). One experimental set consisted of pure aqueous cyclodextrin solutions (Fig. 3A), while the other set contained 0.2 mg/mL hydrocortisone in identical cyclodextrin solutions (Fig. 3B). Some literature data describing osmotic properties of the studied cyclodextrins are available. There is a principal difference between our results and data obtained by Miyajima et al.,27 which observed a decrease in the value of osmotic and activity coefficients with increasing gCD concentration. Our results are similar to those of Terdale et al.28 in case of aCD. However, an algorithm of osmotic coefficients calculation from vapor pressure measurements was not described which makes discussion of possible reasons of such disagreement impossible. In addition, the osmotic studies of HPgCD in a wide concentration range have been carried out by Zannou et al.29 Their JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Figure 3. The experimental osmotic coefficients versus cyclodextrin molality at room temperature ( 238C). Cyclodextrin solutions without hydrocortisone (A); cyclodextrin solutions containing 0.2 mg/mL of hydrocortisone (B). gCD (^); gCD/HPgCD 80/20 (D); gCD/HPgCD 20/80 (); HPgCD (&).
experimental osmolality values for 0–20% (w/v) HPgCD lie between 0 and 200 mOsm/kg that is in agreement with our observations. The activity coefficients were estimated using Eq. (6) and data from Table 4. The concentration dependences of activity coefficients for all studied solutions are shown in Figure 4. The solute activity coefficients increase with concentration showing nonideal behavior of cyclodextrins in the
studied systems (Fig. 4A) which is in agreement with published data for aggregation of aCD.28 The solutions can be arranged according to increasing deviation from ideality: gCD < 80/20 gCD/HPgCD < 20/80 gCD/HPgCD < HPgCD. Such deviations of activity coefficients from unity can be due to either ‘‘solute–solvent’’ interaction, that is, hydration, or ‘‘solute–solute’’ interaction, that is, association or aggregation, or both. The deviations from ideality are here mainly related to the hydration process (namely, to hydrogen-bond formation between oxygen atoms of the solute and water, and hydrogen-bond formation in the peripheral hydration) but the deviation is also related, although to lesser degree, to solute– solute interactions.28,30 The hydration results in increase in the activity coefficients but the aggregation in a decrease. The observed increase in the activity coefficients with increasing solute concentration can be explained as follows. In the aqueous cyclodextrin solutions a competition between hydration and aggregation processes is taking place where hydration prevails over aggregation leading to increase in activity coefficients above unity. Less positive deviation of activity coefficient is observed for pure gCD (Fig. 4A) and, in the context of competition, it means that the ratio between hydration and aggregation terms is shifted in favor of the aggregation in comparison to the other systems. The positive deviation increases with increasing gCD and HPgCD concentrations and the deviation is greater for HPgCD than for the parent gCD. This is because HPgCD is a somewhat larger molecule than gCD which allows for larger number of hydrogen-bonds (i.e., greater hydration) and consequently hydration/aggregation ratio is shifted in favor of the hydration. Regarding solutions containing cyclodextrins and hydrocortisone the solutions can be arranged according
Table 4. Parameters of Eq. (5) for the Studied Aqueous Solutions at Room Temperature ( 238C) Solution gCD 80/20 gCD/HPgCD 20/80 gCD/HPgCD HPgCD gCD þ HC 80/20 gCD/HPgCD þ HC 20/80 gCD/HPgCD þ HC HPgCD þ HC
B1
B2
B3
R
s 102
n
8 1 10 2 15 2 18 1
077 19 104 27 172 33 214 18
270 080 383 121 759 167 995 095
0.9913 0.9902 0.9948 0.9991
1.89 2.55 2.49 1.27
7 7 7 7
7 1 12 4 10 3 11 2
075 27 587 225 140 60 111 39
— 6200 3300 600 300 370 200
0.9858 0.9422 0.9363 0.9878
1.00 1.98 4.43 2.70
6 7 6 6
R, pair correlation coefficient; s, standard deviation; n, number of points. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Figure 4. The estimated activity coefficients of solute versus molality of the cyclodextrins studied in aqueous solutions at room temperature ( 238C). Cyclodextrins without hydrocortisone (A); cyclodextrins with 0.2 mg/mL of hydrocortisone (B). gCD (—); gCD/HPgCD 80/20 (- - -); gCD/HPgCD 20/80 (- - -); HPgCD (. . .).
to increasing deviation from ideality: 80/20 gCD/ HPgCD < gCD < 20/80 gCD/HPgCD < HPgCD. Figure 4B shows that the activity coefficients of all tested drug containing cyclodextrin solutions have in general smaller values than comparable solutions without the drug (Fig. 4A). This is due to the fact that in addition to hydration and aggregation the complexation process takes place in these drug containing solution systems. The complexation causes additional decrease in the value of the activity coefficients. This conclusion is in agreement with complexation data from Table 3 where the 80/20 gCD/HPgCD mixture has the largest CE as well as the largest negative deviation of activity coefficients (Fig. 4B). It would be interesting to determine the driving forces of aggregation process via more detailed thermodynamic analysis that is by estimation of excess thermodynamic parameters of solutes (the Gibbs energy, enthalpy and entropy). This is a matter of our future studies. Transmission electron microscopic (TEM) micrographs of 10% (w/v) gCD and HPgCD solutions, as well as of aqueous 10% (w/v) 80/20 DOI 10.1002/jps
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and 20/80 mixtures of gCD and HPgCD, saturated with hydrocortisone are shown in Figure 5. The sizes of hydrocortisone/gCD and hydrocortisone/ HPgCD complex aggregates are approximately 10–50 and 10–80 nm, respectively (Fig. 5A and B). The aggregate diameter of the hydrocortisone/ (gCD/HPgCD) complex mixtures is much larger than that of gCD and HPgCD or up to couple of hundred nm in diameter, and appeared to be formed by aggregation of smaller complex aggregates (Fig. 5C and D). Previously we have shown that addition of relatively small amounts of the water-soluble HPgCD to aqueous gCD formulations increases the CE of gCD and results in synergistic effect with regard to drug solubilization in aqueous media.20 It is possible that these effects are related to formation of larger aggregates. Other investigators have shown by TEM imaging that the natural a-cyclodextrin (aCD), b-cyclodextrin (bCD) and gCD, and their complexes, form aggregates in pure aqueous solutions.12–14,31 However, these were dilute cyclodextrin solutions (1%, w/v) and the weight fractions of the aggregates were only between 0.0002% and 0.002% of the total cyclodextrin dissolved in the solution.13 Our permeation studies indicate that the weight fraction of the aggregates gradually increases with increasing cyclodextrin concentration, especially when the cyclodextrin concentration exceeds 5% (Figs. 1 and 2). We have made similar observations in aqueous HPbCD solutions.8,15,16
CONCLUSIONS The natural aCD, bCD and gCD, their derivatives, such as HPbCD and HPgCD, and complexes self-assemble to form nanoscale aggregates in aqueous solutions. At cyclodextrin concentration of about 1% (w/v) or lower the relative mass contribution of these aggregates is less than 0.01% but it gradually increases with increasing cyclodextrin concentration until at about 5–10% (w/v) when all increase in dissolved drug/cyclodextrin complexes is in the form of cyclodextrin aggregates. In most cases these solutions do not display static light scattering and appear to the naked eye to be clear solutions and, thus, the diameter of the aggregates is below couple of hundred nanometers. Our studies indicate that most frequently the size is between 10 and 100 nm which is in an agreement with previous studies by other investigators.13,14 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010
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Figure 5. Transmission electron microscopic images of saturated solution of hydrocortisone in 10% cyclodextrin solutions: aggregated hydrocortisone/gCD complexes (A); hydrocortisone/HPgCD complexes (B); hydrocortisone/(gCD/HPgCD) complexes with gCD/HPgCD ratio of 80/20 (C); and hydrocortisone/(gCD/HPgCD) complexes with gCD/HPgCD ratio of 20/80 (D).
ACKNOWLEDGMENTS Financial support from the Eimskip fund, Iceland, is gratefully acknowledged. We gratefully acknowledge the support of Scientific and Technology Research Equipment Centre, Chulalongkorn University, Bangkok, Thailand for TEM analysis.
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