Journal of Colloid and Interface Science 342 (2010) 407–414

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Journal of Colloid and Interface Science

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Colloidal properties of amiodarone in water at low concentration

Luciano Benedini a, Paula V. Messina a, Ruben H. Manzo b, Daniel A. Allemandi b, Santiago D. Palma b,*,Erica P. Schulz a, Marisa A. Frechero a, Pablo C. Schulz a,*

a Departamento de Química e INQUISUR, CONICET, Universidad Nacional del Sur, Bahía Blanca, Argentinab Departamento de Farmacia, Facultad de Ciencias Químicas, CONICET, Universidad Nacional de Córdoba, Argentina

a r t i c l e i n f o

Article history:Received 16 September 2009Accepted 27 October 2009Available online 30 October 2009

Keywords:AmiodaroneCritical micelle concentrationKrafft pointGelCoacervateHydration

0021-9797/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.jcis.2009.10.071

* Corresponding authors.E-mail address: pschulz@criba.edu.ar (P.C. Schulz)

a b s t r a c t

Amiodarone aqueous systems at concentrations C 6 3 mg/mL were studied with different techniques(conductivity, solubility, ion-selective electrodes, pH, viscosity, polarizing microscopy, computationalsimulation, electron microscopy and fluorescence at various temperatures. The Krafft point(TK = 70.6 �C) was determined. A partial phase diagram was plotted showing the critical micelle concen-tration (CMC) dependence on temperature, the solubility and a transition between a coacervate and anunstable gel below TK as a function of temperature. The nature of aggregates below TK at different con-centrations was determined. Micellization is an entropy-driven process which is probably caused by astrong dehydration of the amiodarone backbone on micellization. CMC, micelle aggregation numberand TK given in earlier literature were misinterpreted in the original articles and are re-interpreted in thiswork.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Amiodarone (2-butylbenzofuran-3-yl 4-(2-diethylaminoeth-oxy)-3,5-di-iodophenyl ketone) (Fig. 1) is a class III antiarrhythmicdrug. It is used in the control of ventricular and supraventriculararrhythmias, including arrhythmias associated with Wolff–Parkin-son–White syndrome. It is also indicated for prevention of arrhyth-mias in patients with myocardial infarction or heart failure. Thisdrug is usually administrated by intravenous infusion and closemonitoring of cardiac function is necessary as well as the availabil-ity of resuscitation facilities. Side effects as consequence of amio-darone administration are very common and are dose-related [1].In addition, the excipients that make possible amiodarone solubi-lization in aqueous solution, can also contribute to the appearanceof other kind of side effects such as negative inotropy and hypoten-sion [2,3].

Owing to the low aqueous solubility of amiodarone, injectableformulations require addition of surfactants and co-solvents. So,marketed formulations are compounded with amiodarone hydro-chloride (50 mg/mL), polysorbate 80 (Tween 80, 100 mg/mL) andbenzyl alcohol (20 mg/mL) generally in 3 mL ampuls. Other strate-gies reported in literature proposed the formulation of amiodaronein aqueous media through the dispersion of the hydrochloride salt0.1 mol/L in pH = 3.8 acetate buffer [3–6]. Since the uses of amio-darone are at very low concentrations, knowledge of the colloidal

ll rights reserved.

.

properties at low concentration are fundamental. However, theproperties reported in literature are contradictory. The solubilityof amiodarone at 25 �C was informed to be 0.7 mg/mL and the crit-ical micellar concentration (CMC) was informed as 0.5 mg/mL at25 �C, measured by conductimetric techniques [7,8]. If a high con-centration suspension (50 mg/mL or more) of amiodarone in wateris heated at 60–80 �C, transparent liquid preparations are obtained,which may remain stable at 60 �C [8]. These solutions show com-plex sedimentation profiles during centrifugation, suggesting thepresence of micelles [8]. NMR studies indicated a reversible disrup-tion of aggregates at 60 �C [9]. This clear phase defined as a pseu-do-solution [10] ceased to be transparent and showed a milkyopalescence at room temperature, when diluted at concentrationsclose to the CMC. This behavior is not typical of surfactants close tothe CMC and is an indication that some of the data reported in lit-erature may be misinterpreted. At higher concentrations (far tothose used in pharmaceutical formulations), birefringent lamellarmesophases and a further isotropic state appeared. At 20 �C thepH was almost constant between 20 and 60 mg/mL, and also be-tween 0.5 and 2 mg/mL, but a strong increase appears below0.5 mg/mL [10].

Between 0.5 and 0.75 mg/mL a milky opalescence can be ob-served, suggesting that 0.5 mg/mL cannot be the CMC. At lowerconcentrations the scattering remained high, indicating that thetrue solubility is possibly lower than 0.125 mg/mL. The intensitywas much lower in the 4–50 mg/mL interval, increasing from0.3 � 10�5 to 3.8 � 10�5. The solution was stable between 2 and8 mg/mL with hydrodynamic radius between 100 and 500 nm

Fig. 1. Amiodarone hydrochloride structure.

408 L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414

and high polydispersity, but unstable at about 0.5 mg/mL, withparticle diameters of 10 lm with low polydispersity (100 ± 50nm interval) [10].

Transparent sols and gels were isotropic without any texture inthe polarizing microscope, but show a brief positive flowbirefringence.

The knowledge of the details of self-assembly processes of ami-odarone at low concentration would allow designing a new formu-lation without the necessity of detrimental excipients as explainedabove. In this article we found that some of the data in literaturewere misinterpreted. In particular we found that the Krafft pointof amiodarone in water is higher than that reported in literature,so the transitions below this temperature, interpreted as theCMC in literature cannot really be the critical micelle concentra-tion, and the aggregates detected in the so-called pseudo-solutions[10] are not micelles. We have elucidated the nature of the aggre-gates below and above the Krafft point.

2. Experimental

2.1. Materials

Amiodarone hydrochloride (pharmaceutical grade) was pur-chased from ParafarmTM (Buenos Aires, Argentina). All the reagentswere analytical grade and used without further purification. Redis-tilled water was used in all experiments.

2.2. Conductivity

To determine the critical concentrations at which some struc-tural transition occurs, a mixture of solid Amiodarone and water,with total concentration between 2.5 and 3 mg/mL. The mixturewas heated up to 80 �C and the surfactant was solubilized. The thusobtained solution was allowed to reach the experimental pre-sta-ted temperature. The concentrated solution was titrated withwater at the same temperature, while measuring the specific con-ductivity with an immersion cell and an automatic conductimeter,OAKTON 510 series (Singapore). The specific conductivity (j) wasrepresented as a function of the concentration. The change in theslope of the curve (break point) was indicative of a noticeablechange in aggregation. The conductimeter was calibrated withKCl solutions as usual. Two independent runs were performed, ex-cept for measurements below 70 �C, which were repeated fivetimes to verify the change in the critical concentration dependenceon temperature.

2.3. Solubility measurements

To determine the solubility of amiodarone at different temper-atures, increasing amounts of the drug (0.05, 0.1, 0.2, 1 and 1.8 mg/mL) were suspended in water. Such suspensions were slowlyheated while stirred in a thermostatic water bath. When the

selected temperature was reached, the measurement was per-formed after ten minutes waiting to ensure that the system wasin equilibrium. The specific conductivity was measured and plottedagainst temperature, and the break point (change in the curveslope) was assumed as the temperature in which amiodaronewas totally dissolved. The homogeneous transparent solution ob-served at this point corroborated also this assumption. Measure-ments were repeated twice in independent runs.

2.4. Chloride ion-selective electrode measurements

The activity of chloride ions not included in aggregates wasdetermined at different temperatures (60, 70, 75, 80 and 85 �C)with an Orion Cl� ion-selective electrode against a saturated calo-mel one with a CCT-CONICET-BB Millivoltmeter (Bahia Blanca,Argentina) by titrating the concentrated solution of amiodarone(65 mg in 25 mL water, previously heated to 80 �C to dissolve thesurfactant and then maintained at the working temperature witha water thermostat) with water and measuring the potential. Datawere treated with the usual techniques [11] to obtain the concen-tration of free (non-aggregated) Cl� ions above the aggregationconcentration. The ionization degree (or more strictly, the fractionof chloride ions non-aggregated) was obtained by:

a ¼ 1� ½Cl��agg=½amiodarone�agg ð1Þ

where [Cl�]agg = CT � [Cl�]free is the concentration of chloride ionsbound to aggregates, and [amiodarone]agg = CT � CMC that of amio-darone ions aggregated (in case of T < TK, the CMC is the concentra-tion of the transition). Measurements were repeated twice inindependent runs.

2.5. Aggregation number determination

Steady state fluorescence experiments were carried out with aShimadsu RF-5301 PC Luminiscence Spectrometer (Kyoto, Japan).A 10 nm stoppered rectangular silica cell was placed in a stirredcuvette holder at 30 �C. Both excitation and emission band slitswere fixed at 5 and 1.5 nm respectively and the scan rate selectedat 240 nm/min. Pyrene was used as luminiscence probe and hexa-decylpyridinium chloride C16PyCl was chosen as static quencher.The excitation wavelength was selected at 335 nm, while the emis-sion spectra were collected from 350 to 450 nm. The first and thirdvibronic peaks of the pyrene appear at 373 and 385 nm, respec-tively. The pair pyrene/C16PyCl assures that the residence time ofthe quencher into the micelle is much longer than the fluorescencelifetime of the probe. Solutions were prepared following Infelta andGrätzel procedure [12]. Solutions of amiodarone with C16PyCl andamiodarone with pyrene were prepared at 80 �C and mixed beforemeasurements. The experiments were performed at two times thecritical concentration for all the samples at 30 �C. Pyrene concen-tration was kept constant at around 1 � 10�6 M and the quencherconcentration was varied, depending of the molar fraction of themixed system, from 0 to around 20 � 10�5 M. These values give a[pyrene]/[mixed micelles] and [quencher]/[mixed micelles] ratiosless than 0.01 and 0.9, respectively, assuring a Poisson distribution[13,14]. The following equation is used [14]:

ln I ¼ ln I0 � ½Q �=½micelles� ¼ ln I0 � N½Q �=ðCT � CMCÞ ð2Þ

where [Q] is the concentration of quencher (C16PyCl) and N theaggregation number. Thus plotting ln I vs. [Q] the aggregation num-ber may be obtained from the slope of the linear relationship. Weused the first vibronic peak of pyrene (Imax(I), k = 373 nm). Measure-ments were repeated twice in independent runs.

Table 1Transitions found by conductivity in amiodarone aqueous solutions.

T (�C) T (K) CMC (mg/mL) XCMC

Critical micelle concentration89.8 363 2.22 ± 0.04 5.86E�0584.8 358 1.96 ± 0.03 5.17E�0579.8 353 1.74 ± 0.04 4.59E�0574.8 348 1.69 ± 0.03 4.46E�05

T (�C) T (K) c (mg/mL) x

Transition 269.8 343 1.50 ± 0.03 3.96E�0559.8 333 1.43 ± 0.03 3.78E�0549.8 323 1.2 ± 0.1 3.17E�0539.8 313 1.01 ± 0.03 2.67E�0529.8 303 0.74 ± 0.03 1.95E�05

Table 2Solubility of amiodarone in water at different temperatures.

T (�C) T (K) S (mg/mL) S (mole fraction)

59.6 332.8 0.0436 1.37 � 10�6

62.4 335.5 0.102 3.19 � 10�6

63.1 336.3 0.204 6.37 � 10�6

70.2 343.4 1.00 3.14 � 10�5

70.6 343.7 1.80 5.64 � 10�5

Fig. 2. TEM microphotography of a solution of amiodarone, C = 2.5 mg/mL,T = 25 �C, limpid solution. The bar represents 85 nm. Notice the agglomerations ofsmall spheres surrounded by unorganized small spheres.

L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414 409

2.6. pH measurements

The pH determinations were performed with a millivoltmeterand pH-meter CCT-CONICET-BB and a Broade and James glass elec-trode simultaneously with the chloride ion-selective electrodemeasurements. Commercial buffers with pH = 7.0 and 4.0 wereused for calibration. Measurements were repeated twice in inde-pendent runs.

2.7. Viscosity

Amiodarone was dissolved in tri-distilled water at 80 �C aimingto get a stock solution (6.22 mg/mL) which was then diluted to ob-tain solutions of different concentrations. These dilutions weremaintained at 40, 60 or 80 �C and the viscosity was measured withan Ostwald capillary viscosimeter calibrated with tri-distilledwater. Measurements were repeated twice in independent runs.

2.8. Optical microscopy

Optical microscopy was performed with a Nikon Eclipse E-200POL polarizing (Tokyo, Japan) microscope.

2.9. Transmission electron microscopy (TEM)

TEM was performed following the negative staining techniquewith uranyl acetate using a Jeol 100 CX II 100 kV electron micros-copy (Tokyo, Japan). Details of the procedure were described else-where [15].

2.10. Computational simulation of amiodarone hydration

The simulation of a solvated amiodarone monomer was per-formed by molecular dynamic (MDS) using the AMBER10 [16]molecular simulation suite. After a minimization to adjust the an-gles and bond lengths, the monomer was solvated with 4500 TIP3model waters. First the system was equilibrated at 300 K (Langevinthermostat) and 1 bar and then a dynamic trajectory (canonicalensemble NPT) of 10 ps was done, saving the configurations every0.1 ps. These configurations were analyzed in order to obtain a dis-tance distribution of the water molecules respect the amiodaronesurface.

2.11. Statistics

All least squares fitting of the experimental data where per-formed with the program DataFit 8.1 Oakdale with a confidence le-vel of 0.90. Average values obtained from different experimentswere weighted using the inverse of the variance to obtain the bestminimum variance unbiased average.

3. Results and discussion

Above 70 �C the transition detected by conductivity on dilutionoccurred without changing of the aspect of the solution, which re-mained limpid. At temperatures below 70 �C, the solution becamemilky when diluted below the transition point found by conductiv-ity. We concluded that the transitions above 70 �C corresponded tothe beginning of the process of micellar aggregation of amioda-rone, i.e., the critical micelle concentration (CMC). In other words,Krafft point is TK � 70 �C. Below this temperature, the transitionhas a different nature and was provisionally named Transition 2.Results are shown in Table 1.

The solubility data are tabulated in Table 2. The Krafft temper-ature was determined as TK = 70.6 �C = 343.7 K, from the intersec-

tion between the critical temperature vs. concentration curvedetermined by isothermal dilution and conductivity and the solu-bility vs. concentration curve. At this point, a very remarkable in-crease in solubility was observed. Ravin et al. [8] found a TK

value of 35 �C although the procedure was not very precise andprobably the purity of the surfactant was not as high as that ofour amiodarone. Impurities reduce the Krafft point and inducemicellization.

At temperatures lower than 70.6 �C, the observed transitiondoes not correspond to simple micellar aggregations. These transi-tions produced a change in the appearance of the solutions, whichwas also reported in literature [17]. Moreover, solutions at concen-tration above the transition but below TK cannot be micellar solu-tions, although they are clear. Solutions did not show anybirefringence in the polarizing microscope, both static or underflux. The TEM images showed nearly spherical relatively big aggre-gates (diameter 17.5 ± 0.3 nm) compounded by others smallerspherical aggregates (diameter 2.53 ± 0.05 nm) (see Fig. 2). The

410 L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414

largest aggregates were surrounded by dispersed small spheres.The experiments on pyrene fluorescence gave the data plotted inFig. 3, which allowed us to determine an aggregation numberN = 3.89 ± 0.09. Then, the small aggregates with diameter2.53 ± 0.05 nm may be formed by about four amiodarone mole-cules, aggregated in polydisperse spherical clusters. Notice thatthis is not the micelle aggregation number, since the temperatureof the experiment was below the Krafft point. The only importanceof the preceding information is to give an idea about the coacervatestructure and a possible explanation of the origin of the high poly-dispersity in aggregates found in literature [8]. Since all aggregateswere spherical, the system was not birefringent. The system couldform a cubic liquid crystal, but these mesophases frequently showflow birefringence or birefringence under tensions, whereas thestudied systems did not. Moreover, the system had not the highviscosity frequently associated with cubic mesophases.

Ravin et al. [8] found a micelle aggregation number of about 150at 60 �C by ultracentrifugation. However, this temperature is alsobelow TK. Since Ravin et al. found that these solutions were heter-ogeneous; we believe that they measured not micelles but thepolydisperse clusters of aggregates we found.

Fig. 4 shows the ionization degree of amiodarone aggregates at75 �C and 60 �C determined with Eq. (1). The values at 75 �C (aboveTK) show a dependence on total concentration following therelationship:

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 0.00002 0.00004 0.00006 0.00008 0.0001 0.00012

Q / M

Ln I m

ax(I)

Fig. 3. The logarithm of Imax(I) for pyrene as a function of Q. The slope is �3595 ± 93with R2 = 0.879.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.0025 0.0027 0.0029 0.0031 0.0033 0.0035 0.0037 0.0039 0.0041CT / M

α

Fig. 4. The ionization degree of aggregates at (�) 75 �C and (h) 60 �C as a function ofthe total concentration of amiodarone, determined by ion-selective electrode.

a¼ �ð2:6�2:2Þ�105C2 ðMÞþ ð1:9�1:5Þ�103 CðMÞ�2:7�2:4

R2 ¼ 0:922 ð3Þ

with a confidence level of 0.90. These values are typical of micelles.At 60 �C (below TK) a is very low and almost constant above

C = 0.0032 M (a = 0.079 ± 0.005), while it rises to about 0.8 whenthe concentration is reduced close to the transition zone. This indi-cates that the aggregates above 0.0032 M capture a high propor-tion of Cl� ions, and then the aggregates have a very low charge.This may explain the agglomeration of aggregates, because theelectrostatic repulsion cannot counterbalance the van der Waalsattraction between micelles. Since no crystalline precipitation oc-curred, there must be another constraint. It may be hydrationforces or structural reasons. The almost constant value of a aboveC = 0.0032 M is indicative of a sort of micro-phase separation.

Fig. 5 shows the dependence of pH with concentration for dif-ferent temperatures. It shows that the hydrolysis of amiodaroneat T > TK behaves as the common surfactants which are salts ofweak acids or bases. By extrapolation of the pKa value (obtainedfrom the pH measurements) at 70 �C to

pC = 0, we obtained

pKa = 7.95 ± 0.13, i.e., Ka = (1.13 ± 0.29) � 10�8. This value is com-parable with that of the second ionization of phosphoric acid,6.34 � 10�8 at 25 �C. Above the CMC the hydrolysis continues be-cause the higher solubility in micelles of the neutral form producedby the reaction:

AmHþ þH2O$ AmþH3Oþ

However, when T < TK, there is a hydrolysis almost independentof concentration, which is an indicative of a phase separation.

Measurements of viscosity at 80, 60 and 40 �C were determined(not shown) showing that the viscosity above the critical concen-tration is close to that of the solvent (water) and slightly increaseswhen the concentration is reduced, which is the behavior oppositeto that it is expectable in common micellar solutions. Moreover, at80 �C (above TK) the viscosity below the CMC was reduced as incommon surfactant solutions, but below TK (60 and 40 �C) thereis a slight increase in viscosity, which is an indication of formationof aggregates, not the destruction of them.

Fig. 6 shows a suspension of amiodarone crystals in water, be-fore the system was heated. The system is formed by small acicularcrystals with low birefringence. After heating to 80 �C and cooledto 30 �C the system presented formation of a milky emulsion whichobserved between crossed polaroids (Fig. 7) shows textures typicalof lamellar structures (lamellar mesophases and gel). This material

1.9

2.4

2.9

3.4

3.9

4.4

0 0.001 0.002 0.003 0.004 0.005CT / M

pH

80ºC70ºC60ºC40ºC30ºC

Fig. 5. pH vs. total concentration of amiodarone at different temperatures. Arrowsindicate the critical concentration.

Fig. 6. Acicular crystals of amiodarone suspended in water before heating.

Fig. 7. System amiodarone–water below TK showing textures similar to thosetypical to lamellar mesophases and gel structures. Total concentration 3 mg/mL.Crossed polaroids. 40�.

Fig. 8. Microphotography of amiodarone coagel showing a matrix of gel suspendedin aqueous isotropic liquid, and small amiodarone crystals growing inside the gel.Notice in the left-hand side some textures seeming oily streaks. Total concentration3 mg/mL. Crossed polaroids. 40�.

L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414 411

becomes with time a mixture of globular crystals and gel (which isnamed coagel), as it may be seen in Fig. 8. When the system wasageing by 6 months, the system is formed only by strongly birefrin-gent globular crystals and low birefringent acicular crystals havingan optic axis perpendicular to the needle one. So, when viewedalong this optical axis the crystals are non-birefringent (seeFig. 9a and b).

3.1. The phase diagram

As a conclusion of the above results, the behavior of amiodaronesolutions around the Krafft temperature may be interpreted asfollows.

The Krafft temperature is TK = 70.6 �C, so to achieve a solutionconcentration in water greater than 2 mg/mL of amiodarone, it isnecessary to increase the temperature above this value. After that,

there is a critical concentration which is the CMC. Above the CMCthere is a micellar solution, and below a monomeric solution.

When the micellar solution is cooled below TK, a limpid liquidsolution (which was named ‘‘pseudo-solution” by Ravin et al. [8])is obtained. In the light of our results, this system is really a coac-ervate, i.e., a suspension of one liquid in equilibrium with anotherliquid, both having the same components [18]. The high concentra-tion phase of the coacervate is formed by agglomerates of smallspherical aggregates having about four amiodarone molecules.Since the ionization of the aggregates is low, they remain non-compacted and in solution probably by effect of hydrationconstraints.

When the coacervate is diluted it becomes unstable at a concen-tration in which a coacervate-to-gel transition occurs. This behav-ior was also observed in a coacervate formed in an aqueous sodium10-undecenoate-dodecyltrimethylammonium bromide mixed sys-tem [15]. Gel structure is a lamellar one and shows textures similarto that of lamellar mesophases in the polarizing microscope, whichmay be the cause of confusing the structure formed with this lastphase. However, there are some slight differences between the tex-tures of both phases. As an example, textures in gel phase are coar-ser than in lamellar liquid crystals. By ageing the gel, it becomescoagel (a mixture of gel and crystals) and then crystals. The trans-formation takes several months because it involves structuralchanges in very viscous systems (the suspension of coagel in wateris not very viscous because of the low concentration of the system,but the viscosity inside the coagel particles is very high). As a con-clusion of this study, Fig. 10 shows the partial phase diagram of thesystem around the Krafft transition and the interpretation of itsdifferent regions.

3.2. Thermodynamics of micellization

The CMC data in Fig. 10 were fitted with a quadratic curve andthe minimum was found at T� = 72.6 �C (345.7 K) withx�CMC ¼ 5:32� 10�5 (1.69 mg/mL; 0.00248 M). The plots of CMCvs. temperature for aqueous solutions of surfactants are usuallyU-shaped with a minimum at a characteristic temperature T�

[19]. The ionization degree of aggregates at the transition concen-tration was obtained from a = (@j/@C)M/(@j/@C)m, where (@j/@C)M

and (@j/@C)m are the slopes of the specific conductivity curvesabove and below the critical concentration, respectively, and is

Fig. 9. Crystals of amiodarone obtained after 6 months of ageing, between crossed polaroids: (a) without retardation plate; (b) with retardation selenite plate. The stronglybirefringent globular structures coexist with some low birefringent acicular crystals which viewed perpendicular to their axis are isotropic. For this reason the crystals arevisible only with the retardation plate (b). Total concentration 3 mg/mL.

-14

-13.5

-13

-12.5

-12

-11.5

-11

-10.5

-10

-9.5

-9

300 310 320 330 340 350 360 370T / K

Ln X

criti

c

CMC

KRAFTT TEMPERATURE

GEL ⇔ COAGEL ⇔ CRYSTALS

COACERVATE

MONOMERIC SOLUTION

MICELLAR SOLUTION

SOLUBILITY

COACERVATE ⇔ GEL TRANSITION

Fig. 10. The logarithm of the different critical concentrations (in mole fractionunits) of amiodarone/water system as a function of temperature. The dashed curveis the prolongation of the CMC in a quadratic least squares fitting. This gives aminimum at T = 345.7 K = 72.6 �C with XCMC = 5.318 � 10�5 (1.692 mg/mL), veryclose to the Krafft temperature.

412 L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414

shown in Fig. 11. There is a difference between the a values repre-sented in Fig. 11 and those in Fig. 4 because of the different tech-niques employed. At 60 �C and the critical concentration, the

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

300 310 320 330 340 350 360 370

T / K

α

TK

Fig. 11. Ionization degree of aggregates at the critical concentration as a function oftemperature, determined from conductivity data. The vertical line indicates theKrafft temperature.

value of a obtained from Fig. 4 is 0.11, and from Fig. 11 the valueis 0.169 ± 0.026. At 75 �C, a = 0.60 from Fig. 4, 0.53 ± 0.01 fromFig. 11. Taking into account that the data in Fig. 11 were obtainedfrom conductivity data and those in Fig. 4 from ion-selective elec-trodes, the difference in a values between the two plots is expect-able. However, both plots show the same tendency. It may be seenthat in the micellar zone a rises with the temperature increase asexpected. However, below TK a is very low and becomes almostindependent of T, which is a strong indicative of a change in thestructure of the aggregates, from that of micelles to that of apseudomicrophase .

The thermodynamics of micellization was computed with thefollowing equations:

DGoM ¼ ð1þ aÞRTLnXCMC ð4Þ

DHoM ¼ T2@ðDGo

M=TÞ=@T ð5Þ

DSoM ¼ ðDHo

M � DGoMÞ=T ð6Þ

Results are plotted as a function of the temperature in Fig. 12.By expansion of error, the average error of DGo

M was about 18%.That of DHo

M and DSoM was about 26%. The micellization of amioda-

rone is clearly entropy driven. A large increase in entropy compen-

y = -1.1949x + 380.97R2 = 0.6936

y = 2.1259x - 377.78R2 = 1

y = 3.3208x - 758.76R2 = 0.9445

-100

0

100

200

300

400

500

345 350 355 360 365T / K

ΔGº m

, ΔH

º m, T

ΔSº m

/ kJ

.mol

-1

TΔSºm

ΔHºm

ΔGºm

Fig. 12. Thermodynamic properties of amiodarone micellization as a function oftemperature.

L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414 413

sates the positive enthalpy. Other non-conventional surfactants asbile salts have an entropy-driven micellization [20,21]. In suchcases, the main effect affecting the micellization process is the lib-eration of water molecules caused by the dehydration of surfactantmolecules upon micellization. This means that the amiodarone

Fig. 13. Representation of the hydration of an amiodarone monomer at (a) 3 Å and (b) 5iodine, blue: nitrogen, gray: carbon and white: hydrogen. (For interpretation of the referearticle.)

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5

r

NU

MB

ER O

F W

ATE

R M

OLE

CU

LES

Fig. 14. Histogram showing the number of water molecules in the hydration shel

backbone, having three aromatic rings and three oxygen atoms(Fig. 1), has many hydration water molecules which are liberatedwhen this structure passes from an aqueous environment in mono-mers to a hydrocarbon environment in micelles. The strong inter-action between water and p electrons is well established [22–25]

Å, obtained from AMBER10. Different elements are indicated as: red: oxygen, violet:nces to colour in this figure legend, the reader is referred to the web version of this

6 7 8 9 10

/ Å

l of an amiodarone monomer at different distances, obtained from AMBER10.

414 L. Benedini et al. / Journal of Colloid and Interface Science 342 (2010) 407–414

and may explain this hydration. To verify the possibility that ami-odarone molecules can dehydrate during aggregation, a computa-tional simulation of hydration was performed by using theprogram AMBER10, giving Fig. 13 which are snapshots of a config-uration showing water up to 3 Å (a) and 5 Å (b) from the amioda-rone surface. Fig. 14 shows the number of water molecules as afunction of their distance to the amiodarone monomer surface.This distribution is the average over the 100 configurations ob-tained from the data of the MDS the distribution. From both Figs.13 and 14, we verify that the primary hydration shell of the mole-cule is formed by about 19–20 water molecules, 14 of them apper-taining to the hydrocarbon backbone hydration which are probablyliberated during micellization. Since the hydration of the polarheadgroups is probably retained on aggregation, the existence ofthe hydrocarbon backbone hydration is an important datum.

Since DHoM is a linear function of T, it follows that the change in

heat capacity at constant pressure on micellization DCoP;M is con-

stant. Its value was DCoP;M ¼ ð@DHo

M=@TÞP [19] = 2.13 J/K mol. Thispositive value also supports an increase in degrees of freedomwhen micellization occurs, which may be caused by the liberationof solvent molecules.

4. Conclusions

The Krafft temperature of amiodarone chlorhydrate isTK = 70.6 �C. Above TK, the CMC was found that is an entropy-drivenprocess which is probably caused by a strong dehydration of theamiodarone backbone on micellization. In view of the TK value,the literature transition concentration reported at 25 �C by Ravinet al. [8] is not the CMC. When amiodarone suspensions withC > CMC are heated above TK and then cooled below the Krafftpoint, a coacervate appears, which is formed by agglomerationsof small spherical particles having 2.53 ± 0.05 nm diameter whichin turn agglomerate in larger clusters. The polydisperse aggregatesthat Ravin et al. at 60 �C [8] interpreted as micelles are then ex-plained as clustering of these aggregates. On diluting this coacer-vate becomes unstable and forms a suspension of gel, whichslowly becomes a coagel (i.e., a mixture of gel and crystals) and fi-nally produces crystals.

Commercial formulations of amiodarone produce inconvenientsecondary effects on patients such as injuries in the application siteand further systemic problems. Such effects are mainly produced bythe co-solvents added to solubilize amiodarone [26,27]. Then, abetter knowledge of the amiodarone phase behavior and the ther-modynamics of micellization may aid to the election of the bestco-solvent, i.e., that which can maintain amiodarone in solutionwith the minimum amount of additives, thus reducing adverseeffects.

Acknowledgments

This work was supported by a grant of the Universidad Nationaldel Sur. PVM, DAA, SDP, MAF, EPS and RHM are researchers of theConsejo Nacional de Investigaciones Científicas y Técnicas de laRepública Argentina (CONICET). LB has a fellowship of CONICET.MAF and EPS gratefully acknowledge to AMBER10, University ofCalifornia, San Francisco for kindly authorize the free use of theprogram.

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