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Interaction of amphotericin B with polymeric colloids
Colloids and Surfaces B: Biointerfaces 11 (1998) 141–151
Interaction of amphotericin B with polymeric colloids A spectroscopic study M.S. Espuelas a, P. Legrand b, M. Cheron c, G. Barratt b, F. Puisieux b, J.-Ph. Devissaguet b, J.M. Irache a,* a Departamento de Farmacia y Tecnologı´a Farmace´utica, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain b URA CNRS 1218, Faculte´ de Pharmacie, Universite´ Paris-Sud, 5 av. J.-B. Cle´ment, 92296 Chatenay-Malabry, France c L.P.B.C. (URA CNRS 2056), Universite´ Pierre et Marie Curie, 4 Place Jusssieu, 75252 Paris, Cedex 05, France Received 28 January 1998; accepted 8 April 1998
Abstract The self-association of the amphiphilic antifungal agent amphotericin B (AmB) in water has been shown to depend on numerous parameters which produce spectroscopic changes and is directly related to modifications in AmB toxicity. We have taken advantage of these changes to study the interactions of AmB with two different polymeric materials: poly(e-caprolactone) and poloxamer 188. Both materials are components of a recently developed AmB formulation based on polymeric nanospheres, which have demonstrated reduced acute toxicity in mice as compared with free AmB. Poly(e-caprolactone) composes the core of nanospheres, and poloxamer 188 is the stabilizer used to coat the particles. AmB dispersions with poly(e-caprolactone) nanospheres led to dramatic spectral changes as compared with free AmB dispersions, indicating an extensive reduction of the aggregation state of AmB and an increase in the threshold of its aggregation. These changes seem to be due to the binding of the drug onto the nanosphere surface. In contrast, dispersions of AmB with poloxamer led to an increase of AmB aggregation as a function of poloxamer concentration due to the formation of mixed micelles. Modification of the ionic charge of AmB molecules during the preparation of nanospheres did not have a significant effect on AmB self-organization. However, interaction of AmB with high concentration of poloxamer containing a larger hydrophobic region (polypropylene oxide, PPO) changed this organization by reducing the number of AmB aggregates as compared to the effect of more hydrophilic poloxamers. These results suggest that ionic and/or hydrogen bond interactions only play a minor role and that predominantly hydrophobic forces drive the interaction between AmB and these compounds. Both types of association were easily dissociated upon dilution (AmB concentration<5 mg/ml ), indicating that the interactions of AmB with both polymeric materials are relatively weak. This low affinity between the drug and these compounds has been shown by the fact that such interactions were only achieved with formulations of precise composition obtained with a particular preparative process such as a solvent displacement method. This process forced AmB monomers solubilized in organic solvent — on evaporation of this solvent — to bind to nanospheres or to associate with poloxamer micelles instead of self-aggregating in water. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Amphotericin B; Hydrophobic forces; Micelles; Nanospheres; Poloxamer; Poly(e-caprolactone); Spectroscopy
* Corresponding author. Fax: +48 425649; e-mail: [email protected] 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 03 3 - 2
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1. Introduction Amphotericin B (AmB) is a polyene antibiotic of particular scientific interest. In fact, this molecule has been extensively studied in biophysics as an ionophoric model drug able to form pores in membranes [1] and in medicine as the drug of choice in the treatment of systemic fungal infections [2]. Furthermore, it was one of the first drugs to be incorporated in liposomes, demonstrating the advantages of submicronic colloidal systems [3], and such formulations have recently appeared on the market [4]. Its amphiphilic character facilitated the incorporation of AmB into liposomes or lipid complexes which reduced its toxicity and allowed the administration of higher doses of the antibiotic without severe adverse effects [5]. However, many other systems have been tested as carriers for this antibiotic, including micelles, mixed micelles, emulsions and cyclodextrin complexes (see ref. [4] for a review). Recently, we have developed another AmB carrier, based on poly(ecaprolactone) nanospheres, which is also able to reduce AmB toxicity [6 ]. These nanospheres are formed by a polymeric core of poly(e-caprolactone) coated with a polymeric nonionic surfactant (poloxamer 188) and AmB. Poly(e-caprolactone) is a biodegradable nontoxic hydrophobic polymer used in implants and also proposed as a raw material for the development of nanospheres for ocular or parenteral drug delivery [7]. Poloxamer 188 is a nonionic surfactant which is approved by the FDA as a safe ingredient for injections [8]. According to previous results [6 ], no evidence of antibiotic incorporation within the core of nanospheres was found. Many studies have been devoted to interactions between AmB with micelles of ionic and nonionic surfactant [9,10], with liposomes [11–13] or with lipid complexes [14]. Nevertheless, little is known about the interaction of AmB with polymeric micelles or polymeric nanospheres. Most of these studies have used electronic absorption and circular dichroism spectroscopy. In fact, amphiphilic AmB exhibits a strong absorbance between 300 and 450 nm due to the seven conjugated double bonds of its apolar domain, which is strongly influenced by conformational changes induced by
its self-association in water or by its association with other compounds. The structure of AmB aggregates is very complex and depends on a wide range of parameters related to its pK , ionization a [15] and method of dispersion of the organic stock solution of AmB in an aqueous phase [16 ]. The aim of this work was to study the interactions between AmB and polymeric poly(e-caprolactone) nanospheres. For this purpose, in this first paper we have taken advantage of the spectroscopic properties of AmB to improve our knowledge about its interaction with poloxamer or poly(e-caprolactone). These results will allow us to better understand AmB organization onto spheres prepared with poloxamers in a ternary system which will be described in a second article.
2. Materials and methods 2.1. Reagents Amphotericin B was kindly supplied by Squibb (Madrid, Spain). Poly(e-caprolactone) (MW 42 500), supplied by Sigma (Madrid, Spain) is a biodegradable and biocompatible poly(hydroxy acid ). Poloxamer 188 (Pluronic F68A), poloxamer 184 (Pluronic L64A) and poloxamer 407 (Pluronic F127A) supplied by Comercial Quı´mica Masso´ (Barcelona, Spain) are water soluble nonionic triblock copolymeric surfactants of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) with different molecular weights and different percentages of PPO and PEO ( Table 1). All other chemicals were reagent grade. 2.2. Preparation of suspensions 2.2.1. Amphotericin B nanospheres Amphotericin B nanospheres were prepared by two different procedures. AmB−Nsp — A solvent displacement method described elsewhere [17]. Briefly, AmB (10 mg) and poly(e-caprolactone) (12.5–125 mg) were dissolved in 30 ml of a mixture of organic solvents (methanol:acetone 1:2 v/v), acidified by 0.1 N HCl to solubilize amphotericin B. This organic phase was heated at 50°C for 10 min, and thereafter
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Table 1 Centre [l(e −e =0)] and intensity (De) of the dichroic doublet of AmB in AmB-MM dispersions prepared with different types of l r poloxamers (poloxamer 164, 188 and 407) at two concentrations (1.25 and 12.5 mg/ml ). The table includes the properties of poloxamers [molecular weight and percentage poly(propylene) oxide (PPO) and poly(ethylene) oxide (PEO) region] Molecular weight
Poloxamer 164 Pluronic L64A Poloxamer 188 Pluronic F68A Poloxamer 407 Pluronic F127A
MW PPO region
% PEO region (mass)
2900
1750
40
8400
1750
80
12600
4000
70
[Poloxamer] (mg/ml )
1.25 12.5 1.25 12.5 1.25 12.5
Dichroic doubleta De (M−1 cm−1)
l(e −e =0) (nm) l r
4470 5370 3970 5630 4240 3550
331 330 335 330 329 327.5
a Measurements at 50 mg/ml AmB.
poured into 40 ml of distilled water under moderate magnetic stirring. In order to eliminate the organic solvents, the preparation was evaporated at 55–58°C under vacuum and finally concentrated to 10 ml. Nsp+AmB — Simple mixture of the ingredients, AmB free nanospheres and free AmB. Unloaded nanospheres prepared with 12.5 mg/ml of poloxamer 188 and poly(e-caprolactone) were incubated with an aqueous dispersion of AmB prepared as described later at a concentration of 1 mg/ml overnight.
acetone/methanol solution (AmB-AcM ) of AmB (10 mg) were also prepared in the same way as AmB nanospheres, omitting poloxamer and poly(e-caprolactone), by the solvent displacement method. Aqueous dispersions of AmB obtained from an alkanized acetone/methanol solution (AmBbase/SO) and alkaline dispersion of AmB obtained by pouring the acidified acetone:methanol solution into an aqueous phase with NaOH (AmBbase/AS) were also prepared by the solvent displacement method.
2.2.2. Amphotericin B mixed micelles Amphotericin B mixed micelles (AmB-MM ) of AmB (10 mg) and poloxamer 188 (12.5–125 mg poloxamer) were also prepared in the same way as AmB nanospheres, in absence of poly(e-caprolactone) by the solvent displacement method and also by simple mixing. Mixed micelles of AmB and poloxamer 184 and 407 were designated (AmB-MM184 or AmBMM407) (12.5–125 mg poloxamer).
2.3. Circular dichroism (CD) and UV–visible spectroscopy
2.2.3. Other amphotericin B dispersions AmB dispersions obtained from a mother solution in DMSO (AmB-DMSO) were prepared by solubilization of AmB in DMSO at a concentration of 10 mg/ml followed by dispersion of this organic stock solution in water at a concentration of 1 mg/ml. AmB dispersions obtained from an acidified
UV–visible and CD spectra of AmB-DMSO, AmB-AcM, AmB-MM and AmB−Nsp were recorded on a Cary 1E ( Varian) Mark V dichrograph (Jobin-Yvon). Stock dispersions of antibiotic were diluted with distilled water to final drug concentrations of 50, 10, 5, 1 and 0.5 mg/ml. The pathlengths of the cuvettes were 0.2, 1, 2, 5 and 10 cm respectively. All spectra were recorded at room temperature after equilibrating for at least 30 min. The following parameters were determined: e which is the molar absorption coefficient; De which is the differential molar dichroic absorption coefficient (M−1 cm−1); l(e −e =0) which is the wavelength where the l r two e (e and e ) are equal to zero; left right
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%De/De which is the ratio between the max dichroic doublet of a suspension at a given concentration and the dichroic doublet at the highest concentration. In order to study AmB self-association during the preparation process, UV–visible and CD spectra of the drug were also recorded: (a) after pouring the organic phase into the aqueous phase; (b) after removal of the organic phase, and several times during the concentration of the aqueous phase to a final volume of 10 ml. Drug concentrations in dispersions were also calculated from the absorbance at 405 nm after appropriate dilution in methanol.
3. Results AmB exhibits two very particular physico-chemical properties: amphiphilic behaviour due to the apolar and polar sides of the lactone ring and amphoteric behaviour due to the presence of ionizable carboxyl and amine groups (Fig. 1). As a result of its amphiphilic and zwitterionic nature and the asymmetrical distribution of hydrophobic and hydrophilic groups, the drug is poorly soluble in all aqueous solvents and in many organic solvents where it self-associates and aggregates above a threshold concentration. It is possible to follow the aggregation state of AmB dispersed in aqueous medium by recording electronic absorption and circular dichroism spectra. At concentrations lower than 0.1 mg/ml, amphotericin B is present exclusively in its monomeric form characterized by a peak at 409 nm in both absorption and CD spectra. At higher concentrations, it appears that AmB molecules are
Fig. 1. Amphotericin B structure.
able to self-associate to form oligomers and then aggregates of oligomers. These species have a different spectroscopic behaviour and a bathochromic shift produces a new spectrum with a broad intense single band at 340 nm and other smaller intensity bands at 360, 385 and 420 nm. CD spectra exhibit a very intense negative couplet centred around 340 nm — the intensity of which depends on the AmB concentration — and three negative bands at 368, 393 and 423 nm [18]. Association of amphiphilic AmB with poly(ecaprolactone) nanospheres coated with the polymeric nonionic surfactant poloxamer 188 has recently been shown to influence biological behaviour after I.V. administration in mice [6 ]. Since the toxicity of AmB can be related to its aggregation state [13,16 ], we were interested in the organization of AmB in dispersion with polymeric materials. The effect of their concentration and the method of preparation were analysed. The absorption and circular dichroism spectra of free AmB dispersions, AmB-poloxamer 188 mixed micelles and AmB nanospheres were recorded in order to compare AmB conformational changes within these preparations. 3.1. Influence of type of AmB dispersion 3.1.1. Aqueous dispersions of AmB AmB aqueous dispersions prepared from acidified acetone/methanol solution as described above by complete organic solvent evaporation (AmBAcM ) exhibited a different aggregation state ( Fig. 2) as compared with amphotericin B dispersions prepared by dilution of an aliquot of a DMSO stock solution in water (AmB-DMSO). The dichroic doublet position was blue shifted from 340 to 335 nm. The concentration at which the dichroic doublet began to be observed was slightly increased from 0.1 to 0.5 mg/ml (Fig. 3). Meanwhile, above the critical micellar concentration (CMC ), the intensity of the dichroic doublet increased with increasing AmB concentrations for both AmB-DMSO and AmB-AcM, AmB-AcM was the only AmB dispersion for which the centre of the doublet was progressively blue shifted with the dilution of the AmB stock dispersion (data no shown).
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Fig. 2. Circular dichroism (A) and electronic absorption (B) spectra of AmB dispersions (at 50 mg/ml AmB). (———) AmBAcM; (– – –) AmB-MM[12.5] ([poloxamer]=12.5 mg/ml ); ( ) AmB−Nsp.
Fig. 3. Evolution of dissociation (expressed as %De/e ) of max AmB dispersions: AmB-AcM ( ); AmB–poloxamer 188 (12.5 mg/ml ) mixed micelles (– × –); AmB nanospheres ( ) as a function of dilution.
3.1.2. AmB-poloxamer mixed micelles (Fig. 2) The presence of poloxamer 188 — in the form of 10 nm micelles [19] — either in the aqueous
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Fig. 4. Circular dichroism spectra of AmB-AcM (———) and AmB–poloxamer 188 mixed micelles (AmB-MM ) as a function of poloxamer 188 concentration. [poloxamer 188]=1.25 ( ); 2.5 (– + –); 7.5 (– [ –); 12.5 (———). The AmB concentration was always 1 mg/ml. Dilution for recording spectra at 50 mg/ml (A) and 0.5 mg/ml (B) for AmB.
phase or in the organic phase, during the incorporation of the organic phase containing drug, changed the spectroscopic properties (centre and intensity of the dichroic doublet) of AmB. In fact, the position of the dichroic doublet was progressively blue shifted and its intensity increased when the micelles were prepared with increasing concentrations of poloxamer 188 [Fig. 4(A)]. Dilution of AmB-MM leads to a rapid and extensive dissociation ( Fig. 3). At low dilution (0.5 mg/ml AmB), CD spectra were all centred at the same wavelength, whatever the concentration of poloxamer, suggesting the presence of similar AmB aggregates [Fig. 4(B)]. Electronic absorption spectra showed a slight increase of intensity with increasing poloxamer concentrations as compared with AmB-AcM. The
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band of absorption centred at 335 nm was blue shifted to 332 nm whatever the poloxamer concentration in AmB-MM formulations (data not shown). The incorporation of other poloxamers, poloxamer 184 or 407, instead of poloxamer 188 in the organic phase had the same effect on the dichroic doublet of AmB-AcM: increasing its intensity and blue shift ( Table 1) and in the UV–visible spectra of AmB (increase of intensity without shift of the band at 335 nm for AmB-AcM, data not shown). No differences between them were found when they were incorporated into the formulation at low concentration (1.25 mg/ml ). At high concentration (12.5 mg/ml ), poloxamer 184 and 188 led to the same spectroscopic alterations [ Table 1, $De=5370–5630 and l(e −e =0)=330 nm]. l r However, AmB–poloxamer 407 mixed micelles showed a lower increase of intensity of the dichroic doublet (3550) and its centre was blue shifted (l= 327) as compared to the modifications produced by the other poloxamers. Moreover, poloxamer 407 was the only one which produced a decrease of intensity with increasing concentration (De= 4240 for 1.25 mg/ml as compared to De=3550 for 12.5 mg/ml ).
3.1.3. Amphotericin B nanospheres (Fig. 2) The aggregation state of AmB dispersed in water with poly(e-caprolactone) to form nanospheres in the absence of poloxamer 188 dramatically changed, as showed by CD and absorption spectra, as compared with free AmB dispersions in water. In fact, the CMC of the AmB dispersion was increased from 0.1 to 1 mg/ml. The dichroic doublet appeared above this concentration, but was red shifted from 334 to 346 nm and its intensity decreased 25-fold for the same AmB final concentration [Fig. 2(A)]. Absorption spectra only revealed a wavelength shift of the large band with a slight reduction of intensity [Fig. 2(B)]. Fig. 5 shows the influence of poly(ecaprolactone)/AmB ratio on the evolution of the dichroic doublet of the antibiotic. In fact, an increase of poly(e-caprolactone) concentration in AmB−Nsp dispersions, which proportionally increases the total area of spheres (inner figure),
Fig. 5. Evolution of auto association: centre (– $ –) l(e −e =0) and intensity (De) (– % –) of dichroic doublet of l r AmB nanospheres and area (m2) (inner core) as a function of poly(e-caprolactone)/AmB ratio. The AmB concentration was always 1 mg/ml. Dilution for recording spectra at 50 mg/ml AmB.
was accompanied by an extensive decrease of intensity and a blue shift as compared with the dichroic doublet of self-associated AmB-AcM. The total area of spheres was calculated as follows: Surface (m2/ml )=3W/rr where W is the polymer weight per millilitre, r is the polymer density [1.29×106 g/m3 for poly(ecaprolactone)] and r is the nanosphere radius (m) determined by photon correlation spectroscopy (PCS) on a Zetasizer 4 (Malvern Instruments, UK ). Dilutions of AmB nanospheres led to a similar evolution of intensity to that observed with a simple AmB dispersion or AmB mixed micelles ( Fig. 3). When 0.1 N NaOH was used to solubilize AmB in the organic phase instead of 0.1 N HCl, or when NaOH was added to the aqueous phase before or after the organic phase was poured into the aqueous phase, similar alterations in the spectroscopic properties of AmB to those described above were found (Fig. 6).
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Fig. 6. Circular dichroism spectra of AmB-AcM (———) and AmB nanospheres (at 50 mg/ml for AmB) made as described in methods ( · · · ), with NaOH incorporated in the aqueous phase (AmB-base/AS ) (– × –) or with NaOH incorporated in the organic phase to solubilize AmB (– n –) instead of HCl (AmB-base/SO).
3.2. Influence of the preparation process The evolution of CD spectra of AmB−Nsp preparations as a function of time after pouring the organic phase into the aqueous phase showed that AmB was in its monomeric form before solvent evaporation. Aggregation started only at the end of this evaporation and only small changes in aggregation occurred during the concentration
Fig. 8. Circular dichroism spectra of AmB-AcM (———) and AmB nanospheres prepared by the solvent displacement process (||||||||) or by simple mixing (– ·– · –) of free AmB with unloaded nanospheres. Dilution for recording spectra at 0.5 mg/ml AmB (A) and at 50 mg/ml AmB (B).
Fig. 7. Absorption spectra at different steps during the process of preparation of AmB−Nsp: (1) in the organic solvent (———); (2) in the organic solvent/water mixture [a, after the addition of the organic phase to water ( · · · ); b, after evaporation of the organic phase (– – –); c, after concentration of the dispersion (– × –)].
step ( Fig. 7). A similar evolution was observed for AmB aqueous dispersion and AmB–poloxamer mixed micelles. When a physical mixture of either the drug (as AmB-AcM aqueous dispersion) and poloxamer 188 or the drug and nanospheres was made, spectroscopic modifications due to interactions between AmB and poly(e-caprolactone) or between the drug and poloxamer 188 were observed only at low antibiotic concentrations (<1 mM ) [Fig. 8(A)]. At high concentrations, no differences were found between AmB-AcM and its physical mixture with poloxamer or with nanospheres, even if the contact time between AmB and poloxamer 188 or AmB and polymeric nanospheres was increased from 30 min to overnight [Fig. 8(B)].
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4. Discussion Many authors have been interested in the nature of AmB aggregates formed in water above its critical micellar concentration (CMC ). Therefore, different hypotheses have been proposed based on either results from fitting calculated spectra to experimental ones or from theoretical calculations. Briefly, Ernst et al. [9] were in favour of the presence of a mixture of dimers, oligomers and large aggregates. On the other hand, a double helix structure with a repeated unit containing two AmB molecules lying in the same plane with the polyenic part parallel and facing each other [20] has also been proposed, and a double-length tube-like hydrophobic pore [21]. Recently, the theoretical evaluation of these aggregates by Millie´ et al. [22] led to consideration of the double helix model as the most consistent with the data. It is generally assumed that a change in the dichroic doublet intensity reflects a change in the number of molecules in an aggregated form, and variations of the other spectral characteristics are related to conformational changes of the aggregates (blue or red shifts for the dichroic doublet centre). These conformational changes mainly concern the variation of the distances between centres of neighbouring interacting molecules [23]. Variations in CD spectra of AmB aqueous dispersions (AmB-AcM vs. AmB-DMSO) confirmed the importance of AmB concentration and the nature of the organic stock solution on self-association of the antibiotic in the aqueous phase [16 ]. In this way, alterations in the spectroscopic properties of AmB dispersions (AmB-AcM ) found in the presence of poly(e-caprolactone) or poloxamer can be interpreted as the result of interaction of the drug and polymeric materials. For AmB-MM formulations, the presence of a shift towards the blue region and an increase of intensity of dichroic doublet (as described above) clearly suggests an alteration of the aggregation state of the drug due to an interaction between the drug self-associated in micelles and poloxamer, which was directly related to the initial concentration of poloxamer. These spectroscopic changes have been reported to occur with other surfactants,
such as anionic sodium deoxycholate [9], nonionic lauryl sucrose [10,24], or POE-stearic acid derivatives [25], and they seemed to be correlated with the CMC of surfactants. At CCMC they got AmB as monomers with low ratio AmB/surfactant ratio [24,26 ]. The differences of behaviour between poloxamers and other surfactants could explain the fact that their effect in AmB aggregation state was not correlated with their CMC values. In fact, in our experiments we amply covered the range of these CMC; however, these poloxamers seemed to increase the AmB aggregation state in a certain range of concentrations with high AmB/surfactant ratios. This interaction between the drug and poloxamer could occur through the formation of mixed micelles, in which poloxamer molecules penetrate the amphotericin B micelles. Then, AmB molecules would be closer together inside the aggregated structures and the distance between chromophores reduced, which could explain the blue shift observed in Fig. 2. Moreover, the increase of intensity indicated that the surfactant interacted with AmB aggregates in a way which brought more AmB molecules into AmB mixed micelles. Dilutions of all these micelles resulted in a similar centre of dichroic doublet, revealing a similar, novel organization of AmB. AmB mixed micelles containing different poloxamers showed that poloxamer 407 is the only poloxamer of all the poloxamers tested which was able to reduce the dichroic signal as a function of the concentration of surfactant and reduce AmB self-association. Considering the fact that the only property that differentiates poloxamer 407 from the other poloxamers is its larger PPO domain, this region could participate in the interaction with AmB. Therefore, the interaction between the drug and poloxamer could be driven by mainly hydrophobic forces, as already suggested by Barwicz and coworkers [21,24], Brittain [27], Caillet et al. [28]. Moreover, considering that poloxamer micellization has been reported to involve the PPO region [29], we could imagine interactions between the large PPO domain of the poloxamer and AmB in micelles.
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For AmB−Nsp formulations, the alteration in the aggregation state of AmB associated with the nanospheres seems to be due to the interaction between the drug and poly(e-caprolactone) on the surface of nanospheres. The large reduction of dichroic doublet intensity could be due to fewer stacked AmB aggregates and to a loss of chirality of AmB aggregates symmetrically adsorbed onto spheres, as revealed by the red shift of AmB−Nsp, as compared with AmB aqueous dispersions or AmB-MM. The red shift could be attributed to a novel organization of AmB molecules arranged head-to-tail, with a longer distance between AmB chromophores at the interface [23]. The fact that the reduced AmB self-association is directly related to the increase of area of nanospheres confirms this way of interaction of the drug. Further evidence of this hypothesis has been given by area calculations: thus head-to-tail dimers of AmB molecules at a concentration of 1 mg/ml of drug cover an area sufficient to make a shell around nanospheres (around 0.30 m2/ml is the area required by this number of molecules and this is the area generated by 12.5 mg/ml of polymer when the diameter of nanospheres is about 220 nm). This interaction could occur either by an essentially ionic interaction involving the amino sugar region or by hydrophobic interactions through the double bonds in the chain region (in this case, the ionic head could also play a role). Nevertheless, neutralization of AmB in nanosphere preparations, either in the aqueous phase or in the original organic phase, did not seem to significantly affect the AmB–polymer interaction. This confirms the minimal importance of AmB ionization either during or after the preparation of nanospheres and the predominance of hydrophobic forces between the drug and poly(ecaprolactone). Moreover, it is noteworthy that use of a less hydrophobic polymer such as poly( lactide) instead of the highly hydrophobic poly(e-caprolactone) did not allow the formation of satisfactory AmB nanospheres. The evolution of the intensity of the dichroic doublet of the drug in its self-associated form (free or associated with poloxamer or associated
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to nanospheres) with the dilution has been described as a way of evaluating the stability of this type of interaction [16 ]. Although the aggregates were clearly found to be very different from one another, the decrease of De with the dilution, characteristic of AmB dissociation, was very similar (in terms of percentage of intensity as compared with the maximum intensity). This suggests the existence of a weak interaction between AmB and poloxamer or poly(ecaprolactone) within the mixed micelles or on nanospheres. The results concerning the solvent displacement process indicated that AmB−Nsp organization is evolutive (Fig. 7). In fact, as the polymer precipitated after mixing the organic phase and the aqueous phase, AmB remained soluble in its monomeric form in the mixture. This difference is due to the formation of specific solvent–AmB pairs through the amino sugar region of the antibiotic, which could prevent its self-association, even in the presence of a large amount of water. Brittain [27] and Caillet et al. [28] reported that AmB undergoes self-association into oligomeric species mainly as a result of hydrophobic forces. These hydrophobic interactions are overcome initially by the use of highly polar organic solvents that are also strong donors of electrons pairs and can maintain AmB in the monomeric state. When these solvents are removed by evaporation, these hydrophobic forces are also able to promote interactions with the highly hydrophobic polymer [i.e. poly(caprolactone)] or nonionic surfactants (i.e. poloxamer 188) instead of AmB self-association. Thus, adsorption at the surface of nanospheres and/or formation of mixed micelles occurs. In fact, the driving force of AmB organization in water is a hydrophobic effect. Irrespective of the type of preparation (mixed micelles or nanospheres) and procedure (solvent displacement method or physical mixture of preformed systems), AmB is able to interact with poloxamer within mixed micelles or with poly(e-caprolactone) nanospheres at concentrations lower than its CMC. At concentrations above this value only the first procedure (solvent displacement method) promotes these interactions. This could be due to the fact that the interaction
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is only possible when AmB is in its monomeric form. Once AmB has self-associated, it has no affinity for other compounds. In the case of physical mixture, only monomeric AmB species interact with poly(e-caprolactone) or poloxamer, and for this reason it is only detectable at a low total concentration of drug.
5. Conclusions A direct spectroscopic study was made possible by the presence of distinct signals from the solution, from the mixed micelles and from the surface of nanospheres, which allowed us to observe the following. (1) The formulation and preparation conditions necessary to adsorb the drug onto poly(ecaprolactone) nanospheres or allow interaction with poloxamer 188 are very precise: highly polar organic phase (strong electron pair donor properties) able to achieve a molecular dispersion of AmB in the nanosphere suspensions and hydrophobic polymers able to modify the simple selfassociation of AmB molecules. The effect of the preparative conditions (solvent displacement process, physical mixture of preformed systems) confirms the reported importance of the preparation method on the nature of AmB dispersions, as suggested by Legrand et al. [16 ]. (2) The low affinity between AmB and these compounds also explains the dissociation between AmB–poloxamer and AmB–poly(e-caprolactone) on nanospheres upon dilution. (3) Hydrophobic forces are predominant for both systems. (4) However, the two interactions produced opposing effects on the aggregation state of AmB: the interaction of AmB with the surface of the poly(e-caprolactone) nanospheres promotes small self-aggregates, probably dimers of AmB, whereas AmB–poloxamer interaction increases self-association of the drug.
Acknowledgment The first author thanks the Ministerio de Educacion y Cultura (Spain) for a grant that
enabled her to conduct this research. Special thanks go to Professor J. Bolard for reviewing this manuscript.
References [1] J. Bolard, Biochim. Biophys. Acta 864 (1986) 257–304. [2] R. Janknegt, S. de Marie, A.J.M. Bakker-Woudenberg, D.J.A. Crommelin, Clin. Pharmakin. 23 (1992) 279–281. [3] R.R. New, M.L. Chance, S. Heath, J. Antimicrob. Chemother. 8 (1981) 371–381. [4] J. Brajtburg, J. Bolard, Clin. Microb. Rev. 9 (1996) 512–531. [5] F.C. Szoka, M. Tang, J. Liposome Res. 3 (1993) 363–375. [6 ] M.S. Espuelas, P. Legrand, J.M. Irache, C. Gamazo, A.M. Orecchioni, J.Ph. Devissaguet, P. Ygartua, Int. J. Pharm. 158 (1997) 19–27. [7] V. Masson, F. Maurin, J.P. Devissaguet, H. Fessi, Int. J. Pharm. 139 (1996) 113–123. [8] E. Alleman, R. Gurny, E. Doelker, Eur. J. Pharm. Biopharm. 39 (5) (1993) 173–191. [9] C. Ernst, J. Grange, H. Rinnert, G. Dupont, J. Lematre, Biopolymers 20 (1981) 1575–1588. [10] I. Gruda, D. Milette, M. Brother, G.S. Kobayashi, G. Medoff, J. Brajtburg, Antimicrob. Agents Chemother. 35 (1991) 24–28. [11] A. Vertut-Croquin, J. Bolard, C. Gary-Bobo, Biochem. Biophys. Res. Commun. 125 (1984) 360–365. [12] S. Jullien, A. Vertut-Croquin, J. Brajtbrug, J. Bolard, Anal. Biochem. 172 (1988) 197–202. [13] P. Legrand, M. Che´ron, L. Leroy, J. Bolard, J. Drug Targ. (1997) 311–319. [14] W.R. Perkins, S.R. Minchey, L.T. Boni, C.E. Swenson, M.C. Popescu, R.F. Pasternack, A.S. Janoff, Biochim. Biophys. Acta 1107 (1992) 271–282. [15] J. Mazerski, J. Grzybowska, E. Borowski, J. Eur. Biophys. 18 (1990) 1–6. [16 ] P. Legrand, E.A. Romero, B.E. Cohen, J. Bolard, Antimicrob. Agents Chemother. 36 (1992) 2518–2522. [17] H. Fessi, F. Puisieux, J.Ph. Devissaguet, C. Thies, US Patent 5,118,528 (1987). [18] J. Bolard, M. Seigneuret, G. Boudet, Biochim. Biophys. Acta 599 (1980) 280–293. [19] P. Alexandridis, T.A. Hatton, Colloids Surf. A 96 (1995) 1–46. [20] R.P. Hemenger, T. Kaplan, L.J. Gray, Biopolymers 22 (1983) 911–918. [21] J. Barwicz, W.I. Gruszecki, I. Gruda, J. Colloid Interface Sci. 158 (1993) 71–76. [22] P. Millie´, J. Langlet, J. Berge`s, J. Caillet, J.Ph. Demaret, in preparation. [23] M. Kasha, H.R. Rawls, M.A. El-Bayaumi, Pure Appl. Chem. 11 (1965) 371. [24] J. Barwicz, S. Christian, I. Gruda, Antimicrob. Agents Chemother. 36 (1992) 2310–2315.
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Interaction of amphotericin B with polymeric colloids A spectroscopic study M.S. Espuelas a, P. Legrand b, M. Cheron c, G. Barratt b, F. Puisieux b, J.-Ph. Devissaguet b, J.M. Irache a,* a Departamento de Farmacia y Tecnologı´a Farmace´utica, Facultad de Farmacia, Universidad de Navarra, Pamplona, Spain b URA CNRS 1218, Faculte´ de Pharmacie, Universite´ Paris-Sud, 5 av. J.-B. Cle´ment, 92296 Chatenay-Malabry, France c L.P.B.C. (URA CNRS 2056), Universite´ Pierre et Marie Curie, 4 Place Jusssieu, 75252 Paris, Cedex 05, France Received 28 January 1998; accepted 8 April 1998
Abstract The self-association of the amphiphilic antifungal agent amphotericin B (AmB) in water has been shown to depend on numerous parameters which produce spectroscopic changes and is directly related to modifications in AmB toxicity. We have taken advantage of these changes to study the interactions of AmB with two different polymeric materials: poly(e-caprolactone) and poloxamer 188. Both materials are components of a recently developed AmB formulation based on polymeric nanospheres, which have demonstrated reduced acute toxicity in mice as compared with free AmB. Poly(e-caprolactone) composes the core of nanospheres, and poloxamer 188 is the stabilizer used to coat the particles. AmB dispersions with poly(e-caprolactone) nanospheres led to dramatic spectral changes as compared with free AmB dispersions, indicating an extensive reduction of the aggregation state of AmB and an increase in the threshold of its aggregation. These changes seem to be due to the binding of the drug onto the nanosphere surface. In contrast, dispersions of AmB with poloxamer led to an increase of AmB aggregation as a function of poloxamer concentration due to the formation of mixed micelles. Modification of the ionic charge of AmB molecules during the preparation of nanospheres did not have a significant effect on AmB self-organization. However, interaction of AmB with high concentration of poloxamer containing a larger hydrophobic region (polypropylene oxide, PPO) changed this organization by reducing the number of AmB aggregates as compared to the effect of more hydrophilic poloxamers. These results suggest that ionic and/or hydrogen bond interactions only play a minor role and that predominantly hydrophobic forces drive the interaction between AmB and these compounds. Both types of association were easily dissociated upon dilution (AmB concentration<5 mg/ml ), indicating that the interactions of AmB with both polymeric materials are relatively weak. This low affinity between the drug and these compounds has been shown by the fact that such interactions were only achieved with formulations of precise composition obtained with a particular preparative process such as a solvent displacement method. This process forced AmB monomers solubilized in organic solvent — on evaporation of this solvent — to bind to nanospheres or to associate with poloxamer micelles instead of self-aggregating in water. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Amphotericin B; Hydrophobic forces; Micelles; Nanospheres; Poloxamer; Poly(e-caprolactone); Spectroscopy
* Corresponding author. Fax: +48 425649; e-mail: [email protected] 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 03 3 - 2
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1. Introduction Amphotericin B (AmB) is a polyene antibiotic of particular scientific interest. In fact, this molecule has been extensively studied in biophysics as an ionophoric model drug able to form pores in membranes [1] and in medicine as the drug of choice in the treatment of systemic fungal infections [2]. Furthermore, it was one of the first drugs to be incorporated in liposomes, demonstrating the advantages of submicronic colloidal systems [3], and such formulations have recently appeared on the market [4]. Its amphiphilic character facilitated the incorporation of AmB into liposomes or lipid complexes which reduced its toxicity and allowed the administration of higher doses of the antibiotic without severe adverse effects [5]. However, many other systems have been tested as carriers for this antibiotic, including micelles, mixed micelles, emulsions and cyclodextrin complexes (see ref. [4] for a review). Recently, we have developed another AmB carrier, based on poly(ecaprolactone) nanospheres, which is also able to reduce AmB toxicity [6 ]. These nanospheres are formed by a polymeric core of poly(e-caprolactone) coated with a polymeric nonionic surfactant (poloxamer 188) and AmB. Poly(e-caprolactone) is a biodegradable nontoxic hydrophobic polymer used in implants and also proposed as a raw material for the development of nanospheres for ocular or parenteral drug delivery [7]. Poloxamer 188 is a nonionic surfactant which is approved by the FDA as a safe ingredient for injections [8]. According to previous results [6 ], no evidence of antibiotic incorporation within the core of nanospheres was found. Many studies have been devoted to interactions between AmB with micelles of ionic and nonionic surfactant [9,10], with liposomes [11–13] or with lipid complexes [14]. Nevertheless, little is known about the interaction of AmB with polymeric micelles or polymeric nanospheres. Most of these studies have used electronic absorption and circular dichroism spectroscopy. In fact, amphiphilic AmB exhibits a strong absorbance between 300 and 450 nm due to the seven conjugated double bonds of its apolar domain, which is strongly influenced by conformational changes induced by
its self-association in water or by its association with other compounds. The structure of AmB aggregates is very complex and depends on a wide range of parameters related to its pK , ionization a [15] and method of dispersion of the organic stock solution of AmB in an aqueous phase [16 ]. The aim of this work was to study the interactions between AmB and polymeric poly(e-caprolactone) nanospheres. For this purpose, in this first paper we have taken advantage of the spectroscopic properties of AmB to improve our knowledge about its interaction with poloxamer or poly(e-caprolactone). These results will allow us to better understand AmB organization onto spheres prepared with poloxamers in a ternary system which will be described in a second article.
2. Materials and methods 2.1. Reagents Amphotericin B was kindly supplied by Squibb (Madrid, Spain). Poly(e-caprolactone) (MW 42 500), supplied by Sigma (Madrid, Spain) is a biodegradable and biocompatible poly(hydroxy acid ). Poloxamer 188 (Pluronic F68A), poloxamer 184 (Pluronic L64A) and poloxamer 407 (Pluronic F127A) supplied by Comercial Quı´mica Masso´ (Barcelona, Spain) are water soluble nonionic triblock copolymeric surfactants of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) with different molecular weights and different percentages of PPO and PEO ( Table 1). All other chemicals were reagent grade. 2.2. Preparation of suspensions 2.2.1. Amphotericin B nanospheres Amphotericin B nanospheres were prepared by two different procedures. AmB−Nsp — A solvent displacement method described elsewhere [17]. Briefly, AmB (10 mg) and poly(e-caprolactone) (12.5–125 mg) were dissolved in 30 ml of a mixture of organic solvents (methanol:acetone 1:2 v/v), acidified by 0.1 N HCl to solubilize amphotericin B. This organic phase was heated at 50°C for 10 min, and thereafter
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Table 1 Centre [l(e −e =0)] and intensity (De) of the dichroic doublet of AmB in AmB-MM dispersions prepared with different types of l r poloxamers (poloxamer 164, 188 and 407) at two concentrations (1.25 and 12.5 mg/ml ). The table includes the properties of poloxamers [molecular weight and percentage poly(propylene) oxide (PPO) and poly(ethylene) oxide (PEO) region] Molecular weight
Poloxamer 164 Pluronic L64A Poloxamer 188 Pluronic F68A Poloxamer 407 Pluronic F127A
MW PPO region
% PEO region (mass)
2900
1750
40
8400
1750
80
12600
4000
70
[Poloxamer] (mg/ml )
1.25 12.5 1.25 12.5 1.25 12.5
Dichroic doubleta De (M−1 cm−1)
l(e −e =0) (nm) l r
4470 5370 3970 5630 4240 3550
331 330 335 330 329 327.5
a Measurements at 50 mg/ml AmB.
poured into 40 ml of distilled water under moderate magnetic stirring. In order to eliminate the organic solvents, the preparation was evaporated at 55–58°C under vacuum and finally concentrated to 10 ml. Nsp+AmB — Simple mixture of the ingredients, AmB free nanospheres and free AmB. Unloaded nanospheres prepared with 12.5 mg/ml of poloxamer 188 and poly(e-caprolactone) were incubated with an aqueous dispersion of AmB prepared as described later at a concentration of 1 mg/ml overnight.
acetone/methanol solution (AmB-AcM ) of AmB (10 mg) were also prepared in the same way as AmB nanospheres, omitting poloxamer and poly(e-caprolactone), by the solvent displacement method. Aqueous dispersions of AmB obtained from an alkanized acetone/methanol solution (AmBbase/SO) and alkaline dispersion of AmB obtained by pouring the acidified acetone:methanol solution into an aqueous phase with NaOH (AmBbase/AS) were also prepared by the solvent displacement method.
2.2.2. Amphotericin B mixed micelles Amphotericin B mixed micelles (AmB-MM ) of AmB (10 mg) and poloxamer 188 (12.5–125 mg poloxamer) were also prepared in the same way as AmB nanospheres, in absence of poly(e-caprolactone) by the solvent displacement method and also by simple mixing. Mixed micelles of AmB and poloxamer 184 and 407 were designated (AmB-MM184 or AmBMM407) (12.5–125 mg poloxamer).
2.3. Circular dichroism (CD) and UV–visible spectroscopy
2.2.3. Other amphotericin B dispersions AmB dispersions obtained from a mother solution in DMSO (AmB-DMSO) were prepared by solubilization of AmB in DMSO at a concentration of 10 mg/ml followed by dispersion of this organic stock solution in water at a concentration of 1 mg/ml. AmB dispersions obtained from an acidified
UV–visible and CD spectra of AmB-DMSO, AmB-AcM, AmB-MM and AmB−Nsp were recorded on a Cary 1E ( Varian) Mark V dichrograph (Jobin-Yvon). Stock dispersions of antibiotic were diluted with distilled water to final drug concentrations of 50, 10, 5, 1 and 0.5 mg/ml. The pathlengths of the cuvettes were 0.2, 1, 2, 5 and 10 cm respectively. All spectra were recorded at room temperature after equilibrating for at least 30 min. The following parameters were determined: e which is the molar absorption coefficient; De which is the differential molar dichroic absorption coefficient (M−1 cm−1); l(e −e =0) which is the wavelength where the l r two e (e and e ) are equal to zero; left right
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%De/De which is the ratio between the max dichroic doublet of a suspension at a given concentration and the dichroic doublet at the highest concentration. In order to study AmB self-association during the preparation process, UV–visible and CD spectra of the drug were also recorded: (a) after pouring the organic phase into the aqueous phase; (b) after removal of the organic phase, and several times during the concentration of the aqueous phase to a final volume of 10 ml. Drug concentrations in dispersions were also calculated from the absorbance at 405 nm after appropriate dilution in methanol.
3. Results AmB exhibits two very particular physico-chemical properties: amphiphilic behaviour due to the apolar and polar sides of the lactone ring and amphoteric behaviour due to the presence of ionizable carboxyl and amine groups (Fig. 1). As a result of its amphiphilic and zwitterionic nature and the asymmetrical distribution of hydrophobic and hydrophilic groups, the drug is poorly soluble in all aqueous solvents and in many organic solvents where it self-associates and aggregates above a threshold concentration. It is possible to follow the aggregation state of AmB dispersed in aqueous medium by recording electronic absorption and circular dichroism spectra. At concentrations lower than 0.1 mg/ml, amphotericin B is present exclusively in its monomeric form characterized by a peak at 409 nm in both absorption and CD spectra. At higher concentrations, it appears that AmB molecules are
Fig. 1. Amphotericin B structure.
able to self-associate to form oligomers and then aggregates of oligomers. These species have a different spectroscopic behaviour and a bathochromic shift produces a new spectrum with a broad intense single band at 340 nm and other smaller intensity bands at 360, 385 and 420 nm. CD spectra exhibit a very intense negative couplet centred around 340 nm — the intensity of which depends on the AmB concentration — and three negative bands at 368, 393 and 423 nm [18]. Association of amphiphilic AmB with poly(ecaprolactone) nanospheres coated with the polymeric nonionic surfactant poloxamer 188 has recently been shown to influence biological behaviour after I.V. administration in mice [6 ]. Since the toxicity of AmB can be related to its aggregation state [13,16 ], we were interested in the organization of AmB in dispersion with polymeric materials. The effect of their concentration and the method of preparation were analysed. The absorption and circular dichroism spectra of free AmB dispersions, AmB-poloxamer 188 mixed micelles and AmB nanospheres were recorded in order to compare AmB conformational changes within these preparations. 3.1. Influence of type of AmB dispersion 3.1.1. Aqueous dispersions of AmB AmB aqueous dispersions prepared from acidified acetone/methanol solution as described above by complete organic solvent evaporation (AmBAcM ) exhibited a different aggregation state ( Fig. 2) as compared with amphotericin B dispersions prepared by dilution of an aliquot of a DMSO stock solution in water (AmB-DMSO). The dichroic doublet position was blue shifted from 340 to 335 nm. The concentration at which the dichroic doublet began to be observed was slightly increased from 0.1 to 0.5 mg/ml (Fig. 3). Meanwhile, above the critical micellar concentration (CMC ), the intensity of the dichroic doublet increased with increasing AmB concentrations for both AmB-DMSO and AmB-AcM, AmB-AcM was the only AmB dispersion for which the centre of the doublet was progressively blue shifted with the dilution of the AmB stock dispersion (data no shown).
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Fig. 2. Circular dichroism (A) and electronic absorption (B) spectra of AmB dispersions (at 50 mg/ml AmB). (———) AmBAcM; (– – –) AmB-MM[12.5] ([poloxamer]=12.5 mg/ml ); ( ) AmB−Nsp.
Fig. 3. Evolution of dissociation (expressed as %De/e ) of max AmB dispersions: AmB-AcM ( ); AmB–poloxamer 188 (12.5 mg/ml ) mixed micelles (– × –); AmB nanospheres ( ) as a function of dilution.
3.1.2. AmB-poloxamer mixed micelles (Fig. 2) The presence of poloxamer 188 — in the form of 10 nm micelles [19] — either in the aqueous
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Fig. 4. Circular dichroism spectra of AmB-AcM (———) and AmB–poloxamer 188 mixed micelles (AmB-MM ) as a function of poloxamer 188 concentration. [poloxamer 188]=1.25 ( ); 2.5 (– + –); 7.5 (– [ –); 12.5 (———). The AmB concentration was always 1 mg/ml. Dilution for recording spectra at 50 mg/ml (A) and 0.5 mg/ml (B) for AmB.
phase or in the organic phase, during the incorporation of the organic phase containing drug, changed the spectroscopic properties (centre and intensity of the dichroic doublet) of AmB. In fact, the position of the dichroic doublet was progressively blue shifted and its intensity increased when the micelles were prepared with increasing concentrations of poloxamer 188 [Fig. 4(A)]. Dilution of AmB-MM leads to a rapid and extensive dissociation ( Fig. 3). At low dilution (0.5 mg/ml AmB), CD spectra were all centred at the same wavelength, whatever the concentration of poloxamer, suggesting the presence of similar AmB aggregates [Fig. 4(B)]. Electronic absorption spectra showed a slight increase of intensity with increasing poloxamer concentrations as compared with AmB-AcM. The
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band of absorption centred at 335 nm was blue shifted to 332 nm whatever the poloxamer concentration in AmB-MM formulations (data not shown). The incorporation of other poloxamers, poloxamer 184 or 407, instead of poloxamer 188 in the organic phase had the same effect on the dichroic doublet of AmB-AcM: increasing its intensity and blue shift ( Table 1) and in the UV–visible spectra of AmB (increase of intensity without shift of the band at 335 nm for AmB-AcM, data not shown). No differences between them were found when they were incorporated into the formulation at low concentration (1.25 mg/ml ). At high concentration (12.5 mg/ml ), poloxamer 184 and 188 led to the same spectroscopic alterations [ Table 1, $De=5370–5630 and l(e −e =0)=330 nm]. l r However, AmB–poloxamer 407 mixed micelles showed a lower increase of intensity of the dichroic doublet (3550) and its centre was blue shifted (l= 327) as compared to the modifications produced by the other poloxamers. Moreover, poloxamer 407 was the only one which produced a decrease of intensity with increasing concentration (De= 4240 for 1.25 mg/ml as compared to De=3550 for 12.5 mg/ml ).
3.1.3. Amphotericin B nanospheres (Fig. 2) The aggregation state of AmB dispersed in water with poly(e-caprolactone) to form nanospheres in the absence of poloxamer 188 dramatically changed, as showed by CD and absorption spectra, as compared with free AmB dispersions in water. In fact, the CMC of the AmB dispersion was increased from 0.1 to 1 mg/ml. The dichroic doublet appeared above this concentration, but was red shifted from 334 to 346 nm and its intensity decreased 25-fold for the same AmB final concentration [Fig. 2(A)]. Absorption spectra only revealed a wavelength shift of the large band with a slight reduction of intensity [Fig. 2(B)]. Fig. 5 shows the influence of poly(ecaprolactone)/AmB ratio on the evolution of the dichroic doublet of the antibiotic. In fact, an increase of poly(e-caprolactone) concentration in AmB−Nsp dispersions, which proportionally increases the total area of spheres (inner figure),
Fig. 5. Evolution of auto association: centre (– $ –) l(e −e =0) and intensity (De) (– % –) of dichroic doublet of l r AmB nanospheres and area (m2) (inner core) as a function of poly(e-caprolactone)/AmB ratio. The AmB concentration was always 1 mg/ml. Dilution for recording spectra at 50 mg/ml AmB.
was accompanied by an extensive decrease of intensity and a blue shift as compared with the dichroic doublet of self-associated AmB-AcM. The total area of spheres was calculated as follows: Surface (m2/ml )=3W/rr where W is the polymer weight per millilitre, r is the polymer density [1.29×106 g/m3 for poly(ecaprolactone)] and r is the nanosphere radius (m) determined by photon correlation spectroscopy (PCS) on a Zetasizer 4 (Malvern Instruments, UK ). Dilutions of AmB nanospheres led to a similar evolution of intensity to that observed with a simple AmB dispersion or AmB mixed micelles ( Fig. 3). When 0.1 N NaOH was used to solubilize AmB in the organic phase instead of 0.1 N HCl, or when NaOH was added to the aqueous phase before or after the organic phase was poured into the aqueous phase, similar alterations in the spectroscopic properties of AmB to those described above were found (Fig. 6).
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Fig. 6. Circular dichroism spectra of AmB-AcM (———) and AmB nanospheres (at 50 mg/ml for AmB) made as described in methods ( · · · ), with NaOH incorporated in the aqueous phase (AmB-base/AS ) (– × –) or with NaOH incorporated in the organic phase to solubilize AmB (– n –) instead of HCl (AmB-base/SO).
3.2. Influence of the preparation process The evolution of CD spectra of AmB−Nsp preparations as a function of time after pouring the organic phase into the aqueous phase showed that AmB was in its monomeric form before solvent evaporation. Aggregation started only at the end of this evaporation and only small changes in aggregation occurred during the concentration
Fig. 8. Circular dichroism spectra of AmB-AcM (———) and AmB nanospheres prepared by the solvent displacement process (||||||||) or by simple mixing (– ·– · –) of free AmB with unloaded nanospheres. Dilution for recording spectra at 0.5 mg/ml AmB (A) and at 50 mg/ml AmB (B).
Fig. 7. Absorption spectra at different steps during the process of preparation of AmB−Nsp: (1) in the organic solvent (———); (2) in the organic solvent/water mixture [a, after the addition of the organic phase to water ( · · · ); b, after evaporation of the organic phase (– – –); c, after concentration of the dispersion (– × –)].
step ( Fig. 7). A similar evolution was observed for AmB aqueous dispersion and AmB–poloxamer mixed micelles. When a physical mixture of either the drug (as AmB-AcM aqueous dispersion) and poloxamer 188 or the drug and nanospheres was made, spectroscopic modifications due to interactions between AmB and poly(e-caprolactone) or between the drug and poloxamer 188 were observed only at low antibiotic concentrations (<1 mM ) [Fig. 8(A)]. At high concentrations, no differences were found between AmB-AcM and its physical mixture with poloxamer or with nanospheres, even if the contact time between AmB and poloxamer 188 or AmB and polymeric nanospheres was increased from 30 min to overnight [Fig. 8(B)].
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4. Discussion Many authors have been interested in the nature of AmB aggregates formed in water above its critical micellar concentration (CMC ). Therefore, different hypotheses have been proposed based on either results from fitting calculated spectra to experimental ones or from theoretical calculations. Briefly, Ernst et al. [9] were in favour of the presence of a mixture of dimers, oligomers and large aggregates. On the other hand, a double helix structure with a repeated unit containing two AmB molecules lying in the same plane with the polyenic part parallel and facing each other [20] has also been proposed, and a double-length tube-like hydrophobic pore [21]. Recently, the theoretical evaluation of these aggregates by Millie´ et al. [22] led to consideration of the double helix model as the most consistent with the data. It is generally assumed that a change in the dichroic doublet intensity reflects a change in the number of molecules in an aggregated form, and variations of the other spectral characteristics are related to conformational changes of the aggregates (blue or red shifts for the dichroic doublet centre). These conformational changes mainly concern the variation of the distances between centres of neighbouring interacting molecules [23]. Variations in CD spectra of AmB aqueous dispersions (AmB-AcM vs. AmB-DMSO) confirmed the importance of AmB concentration and the nature of the organic stock solution on self-association of the antibiotic in the aqueous phase [16 ]. In this way, alterations in the spectroscopic properties of AmB dispersions (AmB-AcM ) found in the presence of poly(e-caprolactone) or poloxamer can be interpreted as the result of interaction of the drug and polymeric materials. For AmB-MM formulations, the presence of a shift towards the blue region and an increase of intensity of dichroic doublet (as described above) clearly suggests an alteration of the aggregation state of the drug due to an interaction between the drug self-associated in micelles and poloxamer, which was directly related to the initial concentration of poloxamer. These spectroscopic changes have been reported to occur with other surfactants,
such as anionic sodium deoxycholate [9], nonionic lauryl sucrose [10,24], or POE-stearic acid derivatives [25], and they seemed to be correlated with the CMC of surfactants. At C
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For AmB−Nsp formulations, the alteration in the aggregation state of AmB associated with the nanospheres seems to be due to the interaction between the drug and poly(e-caprolactone) on the surface of nanospheres. The large reduction of dichroic doublet intensity could be due to fewer stacked AmB aggregates and to a loss of chirality of AmB aggregates symmetrically adsorbed onto spheres, as revealed by the red shift of AmB−Nsp, as compared with AmB aqueous dispersions or AmB-MM. The red shift could be attributed to a novel organization of AmB molecules arranged head-to-tail, with a longer distance between AmB chromophores at the interface [23]. The fact that the reduced AmB self-association is directly related to the increase of area of nanospheres confirms this way of interaction of the drug. Further evidence of this hypothesis has been given by area calculations: thus head-to-tail dimers of AmB molecules at a concentration of 1 mg/ml of drug cover an area sufficient to make a shell around nanospheres (around 0.30 m2/ml is the area required by this number of molecules and this is the area generated by 12.5 mg/ml of polymer when the diameter of nanospheres is about 220 nm). This interaction could occur either by an essentially ionic interaction involving the amino sugar region or by hydrophobic interactions through the double bonds in the chain region (in this case, the ionic head could also play a role). Nevertheless, neutralization of AmB in nanosphere preparations, either in the aqueous phase or in the original organic phase, did not seem to significantly affect the AmB–polymer interaction. This confirms the minimal importance of AmB ionization either during or after the preparation of nanospheres and the predominance of hydrophobic forces between the drug and poly(ecaprolactone). Moreover, it is noteworthy that use of a less hydrophobic polymer such as poly( lactide) instead of the highly hydrophobic poly(e-caprolactone) did not allow the formation of satisfactory AmB nanospheres. The evolution of the intensity of the dichroic doublet of the drug in its self-associated form (free or associated with poloxamer or associated
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to nanospheres) with the dilution has been described as a way of evaluating the stability of this type of interaction [16 ]. Although the aggregates were clearly found to be very different from one another, the decrease of De with the dilution, characteristic of AmB dissociation, was very similar (in terms of percentage of intensity as compared with the maximum intensity). This suggests the existence of a weak interaction between AmB and poloxamer or poly(ecaprolactone) within the mixed micelles or on nanospheres. The results concerning the solvent displacement process indicated that AmB−Nsp organization is evolutive (Fig. 7). In fact, as the polymer precipitated after mixing the organic phase and the aqueous phase, AmB remained soluble in its monomeric form in the mixture. This difference is due to the formation of specific solvent–AmB pairs through the amino sugar region of the antibiotic, which could prevent its self-association, even in the presence of a large amount of water. Brittain [27] and Caillet et al. [28] reported that AmB undergoes self-association into oligomeric species mainly as a result of hydrophobic forces. These hydrophobic interactions are overcome initially by the use of highly polar organic solvents that are also strong donors of electrons pairs and can maintain AmB in the monomeric state. When these solvents are removed by evaporation, these hydrophobic forces are also able to promote interactions with the highly hydrophobic polymer [i.e. poly(caprolactone)] or nonionic surfactants (i.e. poloxamer 188) instead of AmB self-association. Thus, adsorption at the surface of nanospheres and/or formation of mixed micelles occurs. In fact, the driving force of AmB organization in water is a hydrophobic effect. Irrespective of the type of preparation (mixed micelles or nanospheres) and procedure (solvent displacement method or physical mixture of preformed systems), AmB is able to interact with poloxamer within mixed micelles or with poly(e-caprolactone) nanospheres at concentrations lower than its CMC. At concentrations above this value only the first procedure (solvent displacement method) promotes these interactions. This could be due to the fact that the interaction
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is only possible when AmB is in its monomeric form. Once AmB has self-associated, it has no affinity for other compounds. In the case of physical mixture, only monomeric AmB species interact with poly(e-caprolactone) or poloxamer, and for this reason it is only detectable at a low total concentration of drug.
5. Conclusions A direct spectroscopic study was made possible by the presence of distinct signals from the solution, from the mixed micelles and from the surface of nanospheres, which allowed us to observe the following. (1) The formulation and preparation conditions necessary to adsorb the drug onto poly(ecaprolactone) nanospheres or allow interaction with poloxamer 188 are very precise: highly polar organic phase (strong electron pair donor properties) able to achieve a molecular dispersion of AmB in the nanosphere suspensions and hydrophobic polymers able to modify the simple selfassociation of AmB molecules. The effect of the preparative conditions (solvent displacement process, physical mixture of preformed systems) confirms the reported importance of the preparation method on the nature of AmB dispersions, as suggested by Legrand et al. [16 ]. (2) The low affinity between AmB and these compounds also explains the dissociation between AmB–poloxamer and AmB–poly(e-caprolactone) on nanospheres upon dilution. (3) Hydrophobic forces are predominant for both systems. (4) However, the two interactions produced opposing effects on the aggregation state of AmB: the interaction of AmB with the surface of the poly(e-caprolactone) nanospheres promotes small self-aggregates, probably dimers of AmB, whereas AmB–poloxamer interaction increases self-association of the drug.
Acknowledgment The first author thanks the Ministerio de Educacion y Cultura (Spain) for a grant that
enabled her to conduct this research. Special thanks go to Professor J. Bolard for reviewing this manuscript.
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