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The ascorbyl palmitate–polyethyleneglycol 400–water system phase behavior
Colloids and Surfaces B: Biointerfaces 89 (2012) 265–270
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
The ascorbyl palmitate–polyethyleneglycol 400–water system phase behavior Luciano Benedini a , Paula V. Messina a , Santiago D. Palma b , Daniel A. Allemandi b , Pablo C. Schulz a,∗ a b
Departamento de Química e INQUISUR, Universidad Nacional del Sur, CONICET, Bahía Blanca, Argentina Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, CONICET, Argentina
a r t i c l e
i n f o
Article history: Received 10 June 2011 Received in revised form 14 September 2011 Accepted 17 September 2011 Available online 22 September 2011 Keywords: Phase diagram Ascorbyl palmitate Liquid crystals Differential scanning calorimetry Polyethyleneglycol 400 (PEG 400)
a b s t r a c t Ascorbyl palmitate (Asc16) in polyethyleneglycol 400 (PEG 400)–water mixtures at weight fractions (w/w) between 0.05 and 1.0 were studied by differential scanning calorimetry (DSC) and polarizing microscopy (PM) at different temperatures. The employed PEG 400–water proportions were: 0–25–50% and 75% of polymer. A complete phase diagram was determined for each PEG 400–water mixture. A cubic mesophase and two (probably three) lamellar mesophases were detected in different regions of the phase diagrams. The addition of PEG 400 to the Asc16–water system shifts the limits of the liquid crystalline domains to lower temperature and surfactant concentration. At weight fraction of PEG 400 ≥ 50%, the limits of the domain of existence of cubic mesophase shift to low surfactant concentration compared with water-rich systems. The hydrated crystals are Asc16.2·6H2 O. If the proportion of water is lower than that value, a mixture of hydrated and anhydrous crystals appears. Heating these crystals produce waxy crystals having melted hydrocarbon bilayers retaining their crystalline structure because the polar bilayers are still rigid. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Palma et al. [1] detected a rich variety of supramolecular assemblies formed by amphiphilic derivatives of ascorbic acid in water. In particular, ascorbyl-6-O-alkanoates possesses three free –OH groups in positions 2 (pKa = 11.6), 3 (pKa = 4.2), and 5 (a secondary hydroxyl group) as shown in Fig. 1, and it was proposed that behaves as anionic surfactants [2]. However, the behavior must be similar to that of aqueous dodecanephosphonic acid, whose pKa values are 2.80 and 8.40 [3] and behaves as a non-ionic surfactant [4,5]. These molecules retain the same radical-scavenging properties of ascorbic acid, and their antioxidant efficiency is comparable to that of other natural reducing agents, such as carotenes, polyphenols, and tocopherols [2]. The large hydrophobic environment of the ASCn coagels seems to be an interesting tool for the loading of drugs with low aqueous solubility. Furthermore, the use of vitamin C based surfactants for micellar solubilization is crucial when the hydrophobic solute is particularly sensitive to light, heat, oxidizing materials and radicals, as the ascorbic polar head groups may provide an efficient shield against these degrading agents, and particularly towards dissolved molecular oxygen. The ascorbyl-6-O-alkanoates–water systems give liquid crystals on heating, which on cooling become gels having lamellar
∗ Corresponding author. Tel.: +54 291 4548305; fax: +54 291 4595160. E-mail address: [email protected] (P.C. Schulz). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.09.030
structure that exhibit sharp X-ray diffraction patterns and optical birefringence. The semisolid consistency of such gels is an interesting property in order to formulate pharmaceutical dosage forms able to solubilise and stabilize different drugs for dermatological use [6], thus obtaining amphiphiles that could combine the powerful antioxidant properties of ascorbic acid with the capacity to produce supramolecular aggregates [6]. Knowledge of the phase behavior of aqueous surfactants is the basis of understanding of the properties of these systems and is vital for the numerous industrial applications of surfactants. The surfactant-water mixtures attract the attention of investigators due to the variability of the phase structures formed by the amphiphilic molecules in water. The rich diversity of phases causes the complexity of the phase equilibriums observed in lyotropic liquid crystalline systems [7]. In a previous work we reported the complete phase diagram of Asc16 (ascorbyl palmitate, 6-O-palmitoyl ascorbic acid, Fig. 2)–water [8]. Below C ≈ 0.48 wt. fraction this system shows hydrated crystals in an isotropic liquid, which give rise to a lamellar liquid crystal when heated to about 60 ◦ C. Above this concentration, the phase transition occurs at about 80 ◦ C giving a cubic liquid crystal which in turn becomes lamellar liquid crystal at about 90 ◦ C. The texture of this lamellar mesophase is different to that produced at lower concentrations. DSC analysis of the melting peaks of water, computer simulation of the hydration of Asc16 molecules and a model of the hydrated aggregates (hydrated crystals and lamellar mesophase) all give the same results: the low concentration
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Fig. 1. Structure of PEG 400.
liquid crystals exist if bulk water is present in the system. There are two other kinds of water other than bulk: hydration water strongly attached by hydrogen bonds to the oxygen and hydrogen atoms of the polar headgroup, which does not melt and is then undetectable by DSC (11.47 ± 0.95 water molecules per surfactant molecule) and the second hydration layer containing 59 ± 17 water molecules per surfactant one (excluding those in the first hydration layer). The number of water molecules in the second hydration layer decreases as the surfactant concentration increases, and vanishes at C = 0.62 ± 0.01 wt. fraction (computational plus structure model data) or at C = 0.667 ± 0.007 (DSC). At very high concentrations the formation of mesophases is preceded by the melt of the chains maintaining the structure of crystals because the anhydrous polar bilayer remains “solid”, giving waxy texture at the polarizing microscope. These findings indicate that the fluid aggregation structures of Asc16 in water appear at temperatures inappropriate to the use in biological media. Then, we intended to modify the limits of the different aggregates domains in the phase diagram by using a non-toxic cosolvent frequently employed in pharmaceutical formulations, polyoxyethyleneglycol 400 (PEG 400, Fig. 1) in different proportions with water. Surfactant and polymer mixed systems form supra-assemblies, which are extensively exploited as active delivery vehicles [9]. These systems include liquid crystalline aggregates (e.g., liposomes and cubosomes) or cross-linked gel networks (hydrogels) that load, stabilize, and eventually deliver active ingredients. Polyethylene glycols (PEGs) are widely used in a variety of pharmaceutical formulations, including parenteral, topical, ophthalmic, oral, and rectal preparations. Polyethylene glycol has been used experimentally in biodegradable polymeric matrices used in controlled-release systems [10] Ambrosi et al. [11] used pharmaceutical cosolvents in low-concentration systems with ascorbyl-6-O-alkanoates, including Asc16, and found that the transition temperature at which different mesophases appear were reduced. According to previous works we observed that the concomitant use of some solvents affected the properties to ASCn coagel systems. In this way, decrease of transition temperature, increase in drug loading capacity and the improvement of drug permeation enhancement of ASCn were observed. The aim of the work is to obtain a system with stable mesophases at room temperature with the goal of use them as drug carriers. These structures are capable of solubilize water-insoluble drugs, and ascorbyl derivatives also increase the skin and mucosa permeability to drugs [6].
Fig. 2. Structure of ascorbyl palmitate (Asc16).
ASCn supramolecular assemblies dissolved hydrophobic drugs such as danthron, phenacetin and griseofulvin in their lipophilic inner cores, and significantly enhance their solubility and availability in the aqueous phase, with respect to pure water [12]. On the other hand, the permeation of ASCn as well as its effect on “in vitro” and “in vivo” drug diffusion through rat skin was evaluated. Penetration of ASCn through rat skin epidermis was very fast and quantitatively significant. Also, the permeation of anthralin from ASCn coagels applied on rat skin was very increased compared to other pharmaceutical systems such as liposomal and niosomal carriers [13] Other active molecules that could be tested to develop a pharmaceutical form based on liquid crystal of this type are clotrimazole and econazole. The first one has similar solubility to Asc16, which is insoluble in water and ethanol soluble. In addition, this molecule is easily degraded and therefore would require the reducing power of Asc16. These molecules have not still been transported in these derivates Ascorbyl coagels. 2. Experimental 2.1. Materials Ascorbyl palmitate (Asc16) was purchased from Flukka (Italy). PEG 400 was purchased from Parafarm (Argentina). All the reagents were analytical grade and used without further purification. Redistilled water by Allchemistry (Buenos Aires, Argentina) was used in all experiments. Solvents were prepared by mixing water and PEG 400 in the proportions 25, 50 and 75 wt.% PEG 400. The samples were prepared by mixing the components (Asc16 and solvent) in the appropriate proportions in closed glass tubes. The dispersions were heated up to 80 ◦ C and then homogenized in an ultrasonic bath for 20 min at ∼60 ◦ C and left to rest in dark for a week days at ∼15 ◦ C in small, hermetically closed plastic tubes before measurements. Samples were prepared having C = 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90 and 0.95 weight/weight (w/w) fraction of surfactant in each solvent. The conditions were chosen to reduce the possibility of Asc16 decomposition by dissolved oxygen [14,15] and the contact with air was strongly reduced in the systems. Moreover, the extent of degradation (in Asc16 sodium salt) is strongly reduced in concentrated systems and in dark. Also, if water is confined, as in W/O microemulsions, the degradation is additionally reduced [16]. Since only the cyclic ring is sensitive to oxidation [16] the extent of degradation in lamellar mesophases and crystals is expected to be additionally reduced in these microstructures because of the very small exposition to water. Moreover, the confinement of water in lamellae reduces the possibility of oxygen diffusion inside the crystalline and liquid crystalline microstructures [16]. 2.2. Differential scanning calorimetry (DSC) measurements Calorimetric measurements were performed with a Q20 Differential Scanning Calorimeter (TA Instruments). Samples were prepared using closed hermetic aluminium pans which have been weighted with a ± 0.00001 g precision balance. The samples were previously cooled to −20 ◦ C during 5 min. Then they were heated up to 150 ◦ C at a rate 5 ◦ C min−1 . Eventually, some samples were kept at this temperature for a minute, to verify if Asc16 decomposition occurs, which might be detected by the observation of heat flow. This situation was not observed, so we concluded that in these conditions (120 ◦ C in hermetic pans) no decomposition occurred. Other samples were cooled at the same rate and then a second run was performed. In these cases, no differences with the first run were detected. Using the transition temperatures the phase diagram for each PEG 400/water system was drawn showing different zones.
L. Benedini et al. / Colloids and Surfaces B: Biointerfaces 89 (2012) 265–270
6
Wt. Fraction of Asc16 0.9
2.7
HEAT FLOW
0.05 Asc16
5
HEAT FLOW
4
0.8
2.6
2.5
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0.7 50
55
60
65
70
0.6
T / ºC
3
0.5 0.4
10
0.3
2
0.2 0.1
1
0
0.05
0
20
40
60
80
100
120
T / ºC Fig. 3. DSC thermograms of Asc16 in PEG 400 25% in water. Inset: amplification of the peak of sample with 0.05 wt. fraction of Asc16.
Superimposed peaks were deconvoluted with the Peakfit program using Gaussian fits. DSC thermograms of the system Asc16 in PEG 400 25%–water are shown in Fig. 3, those of the other systems are not shown because they are similar. 2.3. Optical microscopy Optical microscopy was performed with a Nikon Eclipse E200 POL polarizing microscope (Tokyo, Japan). Samples were put between glass slides on a heating holder and heated to place it in one of the different zones in the phase diagram determined by DSC. Then microphotographs were taken. Also textures appearing on cooling were recorded to determine the various phase changes. Microphotographs were taken with and without crossed polarizers, and in some cases a 1 retardation plate was intercalated. Some representative photomicrographs are shown in Fig. 4. In some of them there are different phases, because there is a temperature gradient between the centre and the edges of the sample on cooling and heating. 3. Results and discussion We used the literature complete phase diagram of Asc16–water system [9] together with some other literature partial data on phase behavior of Asc16/water mixtures [1,17,18] to complete the study. Fig. 5 shows the phase diagrams at different PEG 400/water proportions, together with that in water, obtained from literature [8]. They were obtained by combination of DSC and polarizing microscope measurements. The determination of the nature of the different phases present was done by observation of the polarizing microscope textures at different concentrations and temperatures. The water solubility at 25 ◦ C of Asc16 is 3.46 × 10−7 wt. fraction [18] and no CMC was detected in literature, while the shorter homologous of ascorbyl-6-O-alkanoates series form micelles [18–20]. In a previous paper the Asc16 CMC in water was estimated between 8 × 10−5 M (3.4 × 10−5 wt. fraction) and 1.5 × 10−6 M (6.6 × 10−7 wt. fraction) [8]. In both cases, the solubility at room temperature is well below the estimated CMC. The DSC peak of water melting disappears at a weight proportion of 0.3 Asc16 in the system with 25% of PEG 400 in water. In
the systems having higher polymer proportion the water melting peak was not detected. This may be caused by the hydration of the polymer which eliminates the “bulk” water of the system. In the Asc16–water system, this peak disappears at a proportion of about 0.5 Asc16. No water associated to surfaces was detected in the systems having PEG 400. In the Asc16–water system, below 60 ◦ C and at low concentration, the system is formed by acicular hydrated crystals and an isotropic liquid (dilute Asc16 aqueous solution, Fig. 4A). By increasing the temperature, a two-phase region starts at about 60 ◦ C and finishes at about 70 ◦ C (depending on concentration), giving rise to a lamellar mesophase showing typical low-birefringent smooth oily streaks (Fig. 4B). The same structures can be seen in the systems having PEG 400, but the temperature at which the phase transition begins is lowered as PEG 400 concentration increased, about 60 ◦ C for pure water, about 55 ◦ C for PEG 400 25% in water, about 50 ◦ C for PEG 400 50% in water and about 30 ◦ C for PEG 400 75% in water. Since the texture of more concentrated liquid crystals is different (highly birefringent thick oily streaks with transversal striations, Fig. 4C; and negative spherulites, Fig. 4E), this phase was named low concentration lamellar mesophase and indicated as LCL in the phase diagram. When the temperature increased in Asc16–water systems where no bulk water exists (C ≥ 0.48 wt. fraction), there is a sudden increase of the transition temperature from ∼60 ◦ C to ∼80 ◦ C. Moreover, instead of a single transition from hydrated crystals (CH ) to lamellar mesophase, two transitions occur: from hydrated crystals to a cubic (viscoisotropic, V) mesophase (in the range ∼80 to ∼88 ◦ C) and from the cubic mesophase to a high concentration lamellar liquid crystal (in the range of ∼94 to ∼104 ◦ C, indicated as HCL). This last mesophase has a more birefringent and coarser oily streaks texture. In the PEG 400 25%–water system this phenomenon is also seen at about the same concentration. Since the crystals may incorporate PEG molecules, we have named them solvated crystals (SC). However, when the PEG 400 content is increased (from 25 to 75% in water), the cubic mesophase domain is shifted to lower concentrations. At very high concentration (>0.90 wt. fraction) and above ∼80 ◦ C, crystals showing a waxy texture are seen (Fig. 4G). This texture appears when the polar bilayers of crystals remain “solid” while the hydrocarbon bilayers are “melted” [21]. When chains in
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Fig. 4. Photomicrographs of some of the samples, showing typical textures. Concentrations in weight fraction, crossed polarizers. (A) C = 0.1 in PEG 400 25%–water, 20 ◦ C, showing acicular crystals of Asc16 suspended in the isotropic solution; (B) C = 0.3 in water, 85 ◦ C, showing typical low birefringent oily streaks of low concentration lamellar mesophase (LCL); (C) C = 0.7 in PEG 400 75%–water upon heating, ∼90 ◦ C. High birefringent lamellar liquid crystal (HCL, coarse oily streaks, left), solvent with acicular Asc16 crystals (right). Between crystals and lamellar mesophase, a cubic liquid crystal exists. (D) C = 0.70 in PEG 400 75%–water, upon cooling, showing lamellar liquid crystal (oily streaks, below), cubic mesophase (middle) and gel (above); (E) C = 0.9 in PEG 400 50%–water, 89 ◦ C, showing typical negative spherulites of lamellar liquid crystal and regions of cubic mesophase; (F) C = 0.9 in PEG 400 50%–water, 102 ◦ C on cooling, showing cubic liquid crystal, lamellar mesophase (mosaic) and gel (right) growing from the mesophases; (G) C = 0.95 in PEG 400 25%–water, ∼80 ◦ C, waxy crystals; (H) pure Asc16, 134 ◦ C, 1 retardation plate intercalated, showing cubic liquid crystal, lamellar mosaic texture and some waxy crystals. When the samples are cooled or heated, there is a temperature gradient between the centre and the edges of the slide, which explains the coexistence of several phases.
hydrated crystals melt, the cohesion of the polar network is weakened by hydration and collapses and a liquid crystal forms. But the cohesion of anhydrous polar bilayers is higher and conserves the external crystalline structure when the aliphatic chains “melt”. The transition concentration is almost invariant with PEG 400 concentration. It must be noted that the texture shown by the very high
concentration lamellar mesophase seems that of LCL, i.e., thin low birefringent oily streaks, which may mean that the structure is quite different from that of HCL. The sudden change at C ≈ 0.9 probably means that 10 wt.% of water, about 2.5 water molecules per Asc16 one is the hydration of the amphiphile molecule in crystals; and above 90 wt.% of the
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Fig. 5. The phase diagrams of Asc16 as a function of the composition of solvent. I, isotropic solution; SC, solvated crystals; AC, anhydrous crystals; C, crystals; V, cubic mesophase; LCL, low concentration lamellar liquid crystal; HCL, high concentration lamellar mesophase. The Asc16–water diagram was redrawn from Ref. [8].
surfactant anhydrous molecules appear. This was also verified by extrapolation to zero enthalpy of water melting per gram of sample in the Asc16–water system [8]. When concentrated systems are rapidly cooled from the lamellar liquid crystal to the solid, coarse structures typical of gel phase are seen (Fig. 4D and F). When the system is left at room temperature, it slowly becomes a mixture of isotropic solution and microcrystals. 4. Concluding remarks In this work the whole phase diagrams of three Asc16–water + PEG 400 systems were determined. A cubic mesophase and two (probably three) lamellar mesophases were detected in different regions of the phase diagrams. At very high concentration, waxy crystals having melted hydrocarbon bilayers retaining their crystalline structure (because the polar bilayers are still rigid) are present. The addition of PEG 400 to the Asc16–water system shifts the limits of the liquid crystalline domains to lower temperature and surfactant concentration. When the weight fraction of PEG 400 is equal or above 25%, the limits of the domain of existence of cubic mesophase are shifted to smaller concentration of Asc16 than in water-rich systems. If the proportion of water is lower than 2.5 water molecules per surfactant one, a mixture of hydrated and anhydrous crystals appears, suggesting that the hydrated crystals are Asc16.2·5H2 O. At the higher PEG 400 content explored (75 wt.%), the low concentration lamellar mesophase starts to form at about 30 ◦ C and solvated crystals disappear at about 45 ◦ C. The reduction in the transition temperature to fluid structures is not low enough to employ the system to many pharmaceutical preparations. However, an
Asc16 gel with high PEG 400 may be used to topic applications as proposed in reference [6]. The system is a gel at shelf temperature but by friction on the skin (which is at about 37 ◦ C) the gel will become a liquid crystal. It must be also taken into account that friction also will increase the temperature. Work is in progress to employ a different approach to obtain the desired low-temperature stable fluid aggregates. Acknowledgements This work was supported by a grant of the Universidad Nacional del Sur and other of the Agencia Nacional de Promoción Cientifica y Tecnologica (ANPCyT). PVM and SDP are researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET). LB has a fellowship of CONICET. References [1] S. Palma, A. Jiménez-Kairuz, L. Fratoni, P. Lo Nostro, R. Manzo, D. Allemandi, Il Farmaco 58 (2003) 1271. [2] P.K. Martindale, The Complete Drug Reference, 34th ed., The Pharmaceutical Press, London, 2005. [3] P.C. Schulz, e-Gnosis, 2003, pp. 1–16. www.e-gnosis.udg.mx/revista1. [4] R.M. Minardi, P.C. Schulz, Bruno Vuano, Colloid Polym. Sci. 274 (11) (1996) 1089. [5] R.M. Minardi, P.C. Schulz, B. Vuano, Colloid Polym. Sci. 275 (1997) 754. [6] S.D. Palma, B. Maletto, P. Lo Nostro, R.H. Manzo, M.C. Pistoresi-Palencia, D.A. Allemandi, Drug Dev. Ind. Pharm. 32 (2006) 1. [7] G.G. Chernik, Phase studies of surfactant-water systems, Curr. Opin. Colloid Interface Sci. 4 (2000) 381. [8] L. Benedini, E.P. Schulz, P.V. Messina, S.D. Palma, D.A. Allemandi, P.C. Schulz, Colloids Surf. A: Physcicochem. Eng. Aspects 375 (2011) 178–185. [9] K. Westesen, H. Bunjes, G. Hammer, P.D.A. Siekmann, J. Pharm. Sci. Technol. 55 (4) (2001) 240.
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[10] R.C. Rowe, P.J. Sheskey, M.E. Quinn (Eds.), Handbook of Pharmaceuticals Excipients, 6th ed., Pharmaceutical Press and American Pharmacists Association, London, Chicago, 2009. [11] M. Ambrosi, P. Lo Nostro, L. Fratoni, L. Dei, B.W. Ninham, S. Palma, R.H. Manzo, D. Allemandi, P. Baglioni, Phys. Chem. Chem. Phys. 6 (2004) 1401. [12] S. Palma, R. Manzo, D. Allemandi, L. Fratoni, P. Lo Nostro, Colloids Surf. A: Physicochem. Eng. Aspects 212 (2003) 163. [13] R. Agarwal, O. Katare, S. Vyas, Int. J. Pharm. 228 (2001) 43. [14] K.A. Connors, G.L. Amidon, I.L. Stella, Chemical Stability of Pharmaceuticals, 2nd ed., Wiley – Interscience, New York, 1986.
[15] A.P. Wade, Weller in Handbook of Pharmaceutical Excipients, 2nd ed., American Pharmaceutical Associationm, Washington, DC, 1994, p. 15. [16] P. Spiclin, M. Gasperlin, V. Kmetec, Int. J. Pharm. 222 (2001) 271. [17] P. Lo Nostro, B.W. Ninham, L. Fratoni, S. Palma, R.H. Manzo, D. Allemandi, P. Baglioni, Langmuir 19 (2003) 3222. [18] S. Palma, P. Lo Nostro, R. Manzo, D. Allemandi, Eur. J. Pharm. Sci. 16 (2002) 37. [19] P. Lo Nostro, G. Capuzzi, N. Mulinacci, Langmuir 16 (2000) 1744. [20] P. Lo Nostro, G. Capuzzi, P. Pinelli, N. Mulinacci, A. Romani, F. Vincieri, Colloids Surf. A: Physicochem. Eng. Aspects 167 (2000) 83. [21] P.C. Schulz, M. Abrameto, J.E. Puig, F.A. Soltero-Martínez, A. González-Alvarez, Langmuir 12 (12) (1996) 3082.
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
The ascorbyl palmitate–polyethyleneglycol 400–water system phase behavior Luciano Benedini a , Paula V. Messina a , Santiago D. Palma b , Daniel A. Allemandi b , Pablo C. Schulz a,∗ a b
Departamento de Química e INQUISUR, Universidad Nacional del Sur, CONICET, Bahía Blanca, Argentina Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, CONICET, Argentina
a r t i c l e
i n f o
Article history: Received 10 June 2011 Received in revised form 14 September 2011 Accepted 17 September 2011 Available online 22 September 2011 Keywords: Phase diagram Ascorbyl palmitate Liquid crystals Differential scanning calorimetry Polyethyleneglycol 400 (PEG 400)
a b s t r a c t Ascorbyl palmitate (Asc16) in polyethyleneglycol 400 (PEG 400)–water mixtures at weight fractions (w/w) between 0.05 and 1.0 were studied by differential scanning calorimetry (DSC) and polarizing microscopy (PM) at different temperatures. The employed PEG 400–water proportions were: 0–25–50% and 75% of polymer. A complete phase diagram was determined for each PEG 400–water mixture. A cubic mesophase and two (probably three) lamellar mesophases were detected in different regions of the phase diagrams. The addition of PEG 400 to the Asc16–water system shifts the limits of the liquid crystalline domains to lower temperature and surfactant concentration. At weight fraction of PEG 400 ≥ 50%, the limits of the domain of existence of cubic mesophase shift to low surfactant concentration compared with water-rich systems. The hydrated crystals are Asc16.2·6H2 O. If the proportion of water is lower than that value, a mixture of hydrated and anhydrous crystals appears. Heating these crystals produce waxy crystals having melted hydrocarbon bilayers retaining their crystalline structure because the polar bilayers are still rigid. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Palma et al. [1] detected a rich variety of supramolecular assemblies formed by amphiphilic derivatives of ascorbic acid in water. In particular, ascorbyl-6-O-alkanoates possesses three free –OH groups in positions 2 (pKa = 11.6), 3 (pKa = 4.2), and 5 (a secondary hydroxyl group) as shown in Fig. 1, and it was proposed that behaves as anionic surfactants [2]. However, the behavior must be similar to that of aqueous dodecanephosphonic acid, whose pKa values are 2.80 and 8.40 [3] and behaves as a non-ionic surfactant [4,5]. These molecules retain the same radical-scavenging properties of ascorbic acid, and their antioxidant efficiency is comparable to that of other natural reducing agents, such as carotenes, polyphenols, and tocopherols [2]. The large hydrophobic environment of the ASCn coagels seems to be an interesting tool for the loading of drugs with low aqueous solubility. Furthermore, the use of vitamin C based surfactants for micellar solubilization is crucial when the hydrophobic solute is particularly sensitive to light, heat, oxidizing materials and radicals, as the ascorbic polar head groups may provide an efficient shield against these degrading agents, and particularly towards dissolved molecular oxygen. The ascorbyl-6-O-alkanoates–water systems give liquid crystals on heating, which on cooling become gels having lamellar
∗ Corresponding author. Tel.: +54 291 4548305; fax: +54 291 4595160. E-mail address: [email protected] (P.C. Schulz). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.09.030
structure that exhibit sharp X-ray diffraction patterns and optical birefringence. The semisolid consistency of such gels is an interesting property in order to formulate pharmaceutical dosage forms able to solubilise and stabilize different drugs for dermatological use [6], thus obtaining amphiphiles that could combine the powerful antioxidant properties of ascorbic acid with the capacity to produce supramolecular aggregates [6]. Knowledge of the phase behavior of aqueous surfactants is the basis of understanding of the properties of these systems and is vital for the numerous industrial applications of surfactants. The surfactant-water mixtures attract the attention of investigators due to the variability of the phase structures formed by the amphiphilic molecules in water. The rich diversity of phases causes the complexity of the phase equilibriums observed in lyotropic liquid crystalline systems [7]. In a previous work we reported the complete phase diagram of Asc16 (ascorbyl palmitate, 6-O-palmitoyl ascorbic acid, Fig. 2)–water [8]. Below C ≈ 0.48 wt. fraction this system shows hydrated crystals in an isotropic liquid, which give rise to a lamellar liquid crystal when heated to about 60 ◦ C. Above this concentration, the phase transition occurs at about 80 ◦ C giving a cubic liquid crystal which in turn becomes lamellar liquid crystal at about 90 ◦ C. The texture of this lamellar mesophase is different to that produced at lower concentrations. DSC analysis of the melting peaks of water, computer simulation of the hydration of Asc16 molecules and a model of the hydrated aggregates (hydrated crystals and lamellar mesophase) all give the same results: the low concentration
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Fig. 1. Structure of PEG 400.
liquid crystals exist if bulk water is present in the system. There are two other kinds of water other than bulk: hydration water strongly attached by hydrogen bonds to the oxygen and hydrogen atoms of the polar headgroup, which does not melt and is then undetectable by DSC (11.47 ± 0.95 water molecules per surfactant molecule) and the second hydration layer containing 59 ± 17 water molecules per surfactant one (excluding those in the first hydration layer). The number of water molecules in the second hydration layer decreases as the surfactant concentration increases, and vanishes at C = 0.62 ± 0.01 wt. fraction (computational plus structure model data) or at C = 0.667 ± 0.007 (DSC). At very high concentrations the formation of mesophases is preceded by the melt of the chains maintaining the structure of crystals because the anhydrous polar bilayer remains “solid”, giving waxy texture at the polarizing microscope. These findings indicate that the fluid aggregation structures of Asc16 in water appear at temperatures inappropriate to the use in biological media. Then, we intended to modify the limits of the different aggregates domains in the phase diagram by using a non-toxic cosolvent frequently employed in pharmaceutical formulations, polyoxyethyleneglycol 400 (PEG 400, Fig. 1) in different proportions with water. Surfactant and polymer mixed systems form supra-assemblies, which are extensively exploited as active delivery vehicles [9]. These systems include liquid crystalline aggregates (e.g., liposomes and cubosomes) or cross-linked gel networks (hydrogels) that load, stabilize, and eventually deliver active ingredients. Polyethylene glycols (PEGs) are widely used in a variety of pharmaceutical formulations, including parenteral, topical, ophthalmic, oral, and rectal preparations. Polyethylene glycol has been used experimentally in biodegradable polymeric matrices used in controlled-release systems [10] Ambrosi et al. [11] used pharmaceutical cosolvents in low-concentration systems with ascorbyl-6-O-alkanoates, including Asc16, and found that the transition temperature at which different mesophases appear were reduced. According to previous works we observed that the concomitant use of some solvents affected the properties to ASCn coagel systems. In this way, decrease of transition temperature, increase in drug loading capacity and the improvement of drug permeation enhancement of ASCn were observed. The aim of the work is to obtain a system with stable mesophases at room temperature with the goal of use them as drug carriers. These structures are capable of solubilize water-insoluble drugs, and ascorbyl derivatives also increase the skin and mucosa permeability to drugs [6].
Fig. 2. Structure of ascorbyl palmitate (Asc16).
ASCn supramolecular assemblies dissolved hydrophobic drugs such as danthron, phenacetin and griseofulvin in their lipophilic inner cores, and significantly enhance their solubility and availability in the aqueous phase, with respect to pure water [12]. On the other hand, the permeation of ASCn as well as its effect on “in vitro” and “in vivo” drug diffusion through rat skin was evaluated. Penetration of ASCn through rat skin epidermis was very fast and quantitatively significant. Also, the permeation of anthralin from ASCn coagels applied on rat skin was very increased compared to other pharmaceutical systems such as liposomal and niosomal carriers [13] Other active molecules that could be tested to develop a pharmaceutical form based on liquid crystal of this type are clotrimazole and econazole. The first one has similar solubility to Asc16, which is insoluble in water and ethanol soluble. In addition, this molecule is easily degraded and therefore would require the reducing power of Asc16. These molecules have not still been transported in these derivates Ascorbyl coagels. 2. Experimental 2.1. Materials Ascorbyl palmitate (Asc16) was purchased from Flukka (Italy). PEG 400 was purchased from Parafarm (Argentina). All the reagents were analytical grade and used without further purification. Redistilled water by Allchemistry (Buenos Aires, Argentina) was used in all experiments. Solvents were prepared by mixing water and PEG 400 in the proportions 25, 50 and 75 wt.% PEG 400. The samples were prepared by mixing the components (Asc16 and solvent) in the appropriate proportions in closed glass tubes. The dispersions were heated up to 80 ◦ C and then homogenized in an ultrasonic bath for 20 min at ∼60 ◦ C and left to rest in dark for a week days at ∼15 ◦ C in small, hermetically closed plastic tubes before measurements. Samples were prepared having C = 0.05, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90 and 0.95 weight/weight (w/w) fraction of surfactant in each solvent. The conditions were chosen to reduce the possibility of Asc16 decomposition by dissolved oxygen [14,15] and the contact with air was strongly reduced in the systems. Moreover, the extent of degradation (in Asc16 sodium salt) is strongly reduced in concentrated systems and in dark. Also, if water is confined, as in W/O microemulsions, the degradation is additionally reduced [16]. Since only the cyclic ring is sensitive to oxidation [16] the extent of degradation in lamellar mesophases and crystals is expected to be additionally reduced in these microstructures because of the very small exposition to water. Moreover, the confinement of water in lamellae reduces the possibility of oxygen diffusion inside the crystalline and liquid crystalline microstructures [16]. 2.2. Differential scanning calorimetry (DSC) measurements Calorimetric measurements were performed with a Q20 Differential Scanning Calorimeter (TA Instruments). Samples were prepared using closed hermetic aluminium pans which have been weighted with a ± 0.00001 g precision balance. The samples were previously cooled to −20 ◦ C during 5 min. Then they were heated up to 150 ◦ C at a rate 5 ◦ C min−1 . Eventually, some samples were kept at this temperature for a minute, to verify if Asc16 decomposition occurs, which might be detected by the observation of heat flow. This situation was not observed, so we concluded that in these conditions (120 ◦ C in hermetic pans) no decomposition occurred. Other samples were cooled at the same rate and then a second run was performed. In these cases, no differences with the first run were detected. Using the transition temperatures the phase diagram for each PEG 400/water system was drawn showing different zones.
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6
Wt. Fraction of Asc16 0.9
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HEAT FLOW
4
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2.6
2.5
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0.7 50
55
60
65
70
0.6
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10
0.3
2
0.2 0.1
1
0
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20
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60
80
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T / ºC Fig. 3. DSC thermograms of Asc16 in PEG 400 25% in water. Inset: amplification of the peak of sample with 0.05 wt. fraction of Asc16.
Superimposed peaks were deconvoluted with the Peakfit program using Gaussian fits. DSC thermograms of the system Asc16 in PEG 400 25%–water are shown in Fig. 3, those of the other systems are not shown because they are similar. 2.3. Optical microscopy Optical microscopy was performed with a Nikon Eclipse E200 POL polarizing microscope (Tokyo, Japan). Samples were put between glass slides on a heating holder and heated to place it in one of the different zones in the phase diagram determined by DSC. Then microphotographs were taken. Also textures appearing on cooling were recorded to determine the various phase changes. Microphotographs were taken with and without crossed polarizers, and in some cases a 1 retardation plate was intercalated. Some representative photomicrographs are shown in Fig. 4. In some of them there are different phases, because there is a temperature gradient between the centre and the edges of the sample on cooling and heating. 3. Results and discussion We used the literature complete phase diagram of Asc16–water system [9] together with some other literature partial data on phase behavior of Asc16/water mixtures [1,17,18] to complete the study. Fig. 5 shows the phase diagrams at different PEG 400/water proportions, together with that in water, obtained from literature [8]. They were obtained by combination of DSC and polarizing microscope measurements. The determination of the nature of the different phases present was done by observation of the polarizing microscope textures at different concentrations and temperatures. The water solubility at 25 ◦ C of Asc16 is 3.46 × 10−7 wt. fraction [18] and no CMC was detected in literature, while the shorter homologous of ascorbyl-6-O-alkanoates series form micelles [18–20]. In a previous paper the Asc16 CMC in water was estimated between 8 × 10−5 M (3.4 × 10−5 wt. fraction) and 1.5 × 10−6 M (6.6 × 10−7 wt. fraction) [8]. In both cases, the solubility at room temperature is well below the estimated CMC. The DSC peak of water melting disappears at a weight proportion of 0.3 Asc16 in the system with 25% of PEG 400 in water. In
the systems having higher polymer proportion the water melting peak was not detected. This may be caused by the hydration of the polymer which eliminates the “bulk” water of the system. In the Asc16–water system, this peak disappears at a proportion of about 0.5 Asc16. No water associated to surfaces was detected in the systems having PEG 400. In the Asc16–water system, below 60 ◦ C and at low concentration, the system is formed by acicular hydrated crystals and an isotropic liquid (dilute Asc16 aqueous solution, Fig. 4A). By increasing the temperature, a two-phase region starts at about 60 ◦ C and finishes at about 70 ◦ C (depending on concentration), giving rise to a lamellar mesophase showing typical low-birefringent smooth oily streaks (Fig. 4B). The same structures can be seen in the systems having PEG 400, but the temperature at which the phase transition begins is lowered as PEG 400 concentration increased, about 60 ◦ C for pure water, about 55 ◦ C for PEG 400 25% in water, about 50 ◦ C for PEG 400 50% in water and about 30 ◦ C for PEG 400 75% in water. Since the texture of more concentrated liquid crystals is different (highly birefringent thick oily streaks with transversal striations, Fig. 4C; and negative spherulites, Fig. 4E), this phase was named low concentration lamellar mesophase and indicated as LCL in the phase diagram. When the temperature increased in Asc16–water systems where no bulk water exists (C ≥ 0.48 wt. fraction), there is a sudden increase of the transition temperature from ∼60 ◦ C to ∼80 ◦ C. Moreover, instead of a single transition from hydrated crystals (CH ) to lamellar mesophase, two transitions occur: from hydrated crystals to a cubic (viscoisotropic, V) mesophase (in the range ∼80 to ∼88 ◦ C) and from the cubic mesophase to a high concentration lamellar liquid crystal (in the range of ∼94 to ∼104 ◦ C, indicated as HCL). This last mesophase has a more birefringent and coarser oily streaks texture. In the PEG 400 25%–water system this phenomenon is also seen at about the same concentration. Since the crystals may incorporate PEG molecules, we have named them solvated crystals (SC). However, when the PEG 400 content is increased (from 25 to 75% in water), the cubic mesophase domain is shifted to lower concentrations. At very high concentration (>0.90 wt. fraction) and above ∼80 ◦ C, crystals showing a waxy texture are seen (Fig. 4G). This texture appears when the polar bilayers of crystals remain “solid” while the hydrocarbon bilayers are “melted” [21]. When chains in
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Fig. 4. Photomicrographs of some of the samples, showing typical textures. Concentrations in weight fraction, crossed polarizers. (A) C = 0.1 in PEG 400 25%–water, 20 ◦ C, showing acicular crystals of Asc16 suspended in the isotropic solution; (B) C = 0.3 in water, 85 ◦ C, showing typical low birefringent oily streaks of low concentration lamellar mesophase (LCL); (C) C = 0.7 in PEG 400 75%–water upon heating, ∼90 ◦ C. High birefringent lamellar liquid crystal (HCL, coarse oily streaks, left), solvent with acicular Asc16 crystals (right). Between crystals and lamellar mesophase, a cubic liquid crystal exists. (D) C = 0.70 in PEG 400 75%–water, upon cooling, showing lamellar liquid crystal (oily streaks, below), cubic mesophase (middle) and gel (above); (E) C = 0.9 in PEG 400 50%–water, 89 ◦ C, showing typical negative spherulites of lamellar liquid crystal and regions of cubic mesophase; (F) C = 0.9 in PEG 400 50%–water, 102 ◦ C on cooling, showing cubic liquid crystal, lamellar mesophase (mosaic) and gel (right) growing from the mesophases; (G) C = 0.95 in PEG 400 25%–water, ∼80 ◦ C, waxy crystals; (H) pure Asc16, 134 ◦ C, 1 retardation plate intercalated, showing cubic liquid crystal, lamellar mosaic texture and some waxy crystals. When the samples are cooled or heated, there is a temperature gradient between the centre and the edges of the slide, which explains the coexistence of several phases.
hydrated crystals melt, the cohesion of the polar network is weakened by hydration and collapses and a liquid crystal forms. But the cohesion of anhydrous polar bilayers is higher and conserves the external crystalline structure when the aliphatic chains “melt”. The transition concentration is almost invariant with PEG 400 concentration. It must be noted that the texture shown by the very high
concentration lamellar mesophase seems that of LCL, i.e., thin low birefringent oily streaks, which may mean that the structure is quite different from that of HCL. The sudden change at C ≈ 0.9 probably means that 10 wt.% of water, about 2.5 water molecules per Asc16 one is the hydration of the amphiphile molecule in crystals; and above 90 wt.% of the
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Fig. 5. The phase diagrams of Asc16 as a function of the composition of solvent. I, isotropic solution; SC, solvated crystals; AC, anhydrous crystals; C, crystals; V, cubic mesophase; LCL, low concentration lamellar liquid crystal; HCL, high concentration lamellar mesophase. The Asc16–water diagram was redrawn from Ref. [8].
surfactant anhydrous molecules appear. This was also verified by extrapolation to zero enthalpy of water melting per gram of sample in the Asc16–water system [8]. When concentrated systems are rapidly cooled from the lamellar liquid crystal to the solid, coarse structures typical of gel phase are seen (Fig. 4D and F). When the system is left at room temperature, it slowly becomes a mixture of isotropic solution and microcrystals. 4. Concluding remarks In this work the whole phase diagrams of three Asc16–water + PEG 400 systems were determined. A cubic mesophase and two (probably three) lamellar mesophases were detected in different regions of the phase diagrams. At very high concentration, waxy crystals having melted hydrocarbon bilayers retaining their crystalline structure (because the polar bilayers are still rigid) are present. The addition of PEG 400 to the Asc16–water system shifts the limits of the liquid crystalline domains to lower temperature and surfactant concentration. When the weight fraction of PEG 400 is equal or above 25%, the limits of the domain of existence of cubic mesophase are shifted to smaller concentration of Asc16 than in water-rich systems. If the proportion of water is lower than 2.5 water molecules per surfactant one, a mixture of hydrated and anhydrous crystals appears, suggesting that the hydrated crystals are Asc16.2·5H2 O. At the higher PEG 400 content explored (75 wt.%), the low concentration lamellar mesophase starts to form at about 30 ◦ C and solvated crystals disappear at about 45 ◦ C. The reduction in the transition temperature to fluid structures is not low enough to employ the system to many pharmaceutical preparations. However, an
Asc16 gel with high PEG 400 may be used to topic applications as proposed in reference [6]. The system is a gel at shelf temperature but by friction on the skin (which is at about 37 ◦ C) the gel will become a liquid crystal. It must be also taken into account that friction also will increase the temperature. Work is in progress to employ a different approach to obtain the desired low-temperature stable fluid aggregates. Acknowledgements This work was supported by a grant of the Universidad Nacional del Sur and other of the Agencia Nacional de Promoción Cientifica y Tecnologica (ANPCyT). PVM and SDP are researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET). LB has a fellowship of CONICET. References [1] S. Palma, A. Jiménez-Kairuz, L. Fratoni, P. Lo Nostro, R. Manzo, D. Allemandi, Il Farmaco 58 (2003) 1271. [2] P.K. Martindale, The Complete Drug Reference, 34th ed., The Pharmaceutical Press, London, 2005. [3] P.C. Schulz, e-Gnosis, 2003, pp. 1–16. www.e-gnosis.udg.mx/revista1. [4] R.M. Minardi, P.C. Schulz, Bruno Vuano, Colloid Polym. Sci. 274 (11) (1996) 1089. [5] R.M. Minardi, P.C. Schulz, B. Vuano, Colloid Polym. Sci. 275 (1997) 754. [6] S.D. Palma, B. Maletto, P. Lo Nostro, R.H. Manzo, M.C. Pistoresi-Palencia, D.A. Allemandi, Drug Dev. Ind. Pharm. 32 (2006) 1. [7] G.G. Chernik, Phase studies of surfactant-water systems, Curr. Opin. Colloid Interface Sci. 4 (2000) 381. [8] L. Benedini, E.P. Schulz, P.V. Messina, S.D. Palma, D.A. Allemandi, P.C. Schulz, Colloids Surf. A: Physcicochem. Eng. Aspects 375 (2011) 178–185. [9] K. Westesen, H. Bunjes, G. Hammer, P.D.A. Siekmann, J. Pharm. Sci. Technol. 55 (4) (2001) 240.
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