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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery systems
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery systems K. Mohsin1, 2*, C.W. Pouton1 1 Monash Institute of Pharmaceutical Sciences, Monash University, Australia Present address: Kayyali Chair for Pharmaceutical Industries, KSU, Riyadh, Saudi Arabia *Correspondence: [email protected]
2
The study was designed to investigate phase behavior during dispersion of anhydrous lipid formulations, and to investigate the effect of lipid/ surfactant ratios, and the presence of cosolvent on the performance of formulations. Equilibrium phase studies were extensively conducted using three component systems to investigate the phase changes of the anhydrous formulation in response to aqueous dilution. The influence of chain length and combination of mono-, di- and tri-glycerides on the phase behavior was studied. Droplet size studies were carried out to assess the influence of lipid and surfactant on the resultant droplet size upon aqueous dilution. The study shows that mixture of mono-, di- and tri-glycerides with equal amount of surfactant can promote great absorption of water and produce efficient self-emulsification systems (particularly, self-emulsifying/ microemulsifying drug delivery systems). Increasing the monoglyceride concentration within the oil component enhanced water solubilization significantly. Among the phase diagram studies, Imwitor 308/Tween 80 systems produced a large optically transparent nanodispersing region. The selection of glycerides seems to be the most vital oil component in designing optimal self-emulsifying lipid formulations. Key words: Lipid-based formulation – Self-emulsifying systems – Ternary phase diagram – Particle size analysis. From the last decade, there has been increasing focus on the utility of self-emulsifying/microemulsifying drug delivery systems (SEDDS/ SMEDDS) to enhance the oral bioavailability of large number lipophilic drugs [1, 2]. It has been well documented that SEDDS/ SMEDDS can be formulated with various lipid excipients, which are homogenous mixtures of natural or synthetic oils, water-soluble or insoluble surfactants or alternatively, one or more hydrophilic solvents and cosolvents [3]. The materials (i.e. vegetable oil derivatives, nonionic surfactants etc.) used in the systems are suitable for oral ingestion [4-6]. SEDDS/SMEDDS formulations are either water free or low in water content which allows encapsulation of a final unit dose in soft or hard gelatin. Thus they help to minimize irritation caused by the regular solid bulk drug substances with the gut wall. A number of early studies have shown that [7] only very specific combination of pharmaceutical excipients led to efficient self-emulsifying systems, which expected to self-emulsify rapidly in the aqueous contents of the stomach. As a result, it presents the drug in fine droplets of oil (< 5 µm) without any precipitation. Fine oil droplets should empty rapidly from the stomach and promote faster and immediate drug release throughout the GI tract. The principal characteristic of these systems is their ability to form fine oil in water (o/w) emulsions or mostly a single phase microemulsions (oil or water continuous phase) upon mild agitation when exposed to aqueous phases. It has a particular relevance in the understanding and the control of the formulation and stability of preparations in which water and oil coexist, such as dispersed systems [8, 9]. It is well known that aqueous mass transfers are likely to occur between the dispersed drops, through the oil continuous phase. These transfers are probably highly influenced by surfactants, which are necessarily present in the emulsions, and that give sometimes direct or reverse micelles, inside which the aqueous or oily transport will be made easier. Indeed, SMEDDS in another term “microemulsions” are thermodynamically stable, macroscopically isotropic and nanostructured mixtures, which often require high surfactant concentrations in order to microemulsify entire oil and water phase [10]. Single phase microemulsions are currently of interest to the pharmaceutical scientist as potential drug delivery vehicles due to their long term stability, ease of preparation, and considerable capacity for solubilization of a variety of drug molecules [11, 12].
It is worth mentioning that if a hydrophobic drug has adequate solubility in oil or oil/surfactant blends then SEDDS/SMEDDS can be a great option for their oral delivery [13]. According to many reports, medium chain fatty acids, mono-/di-, and tri-glycerides, particularly caprylic/capric mono-/di-glycerides have been used independently in mixed micelle emulsion, microemulsion and SEDDS/SMEDDS formulations and as absorption enhancers for a number of drugs, since they have been found to be physiologically well tolerated [14]. It is important for the current studies to note that glycerides are the oil component of the formulations. Within the scope of the present studies, it was required majorly to identify the self-emulsifying formulations using wide ranges of oils composed of medium chain mono-, di-, and tri-glycerides, water-soluble cosolvents and commonly used non-ionic surfactants following the lipid formulation classification system (LFCS) [15]. To fulfill this requirement, a series of phase diagrams were constructed to study the equilibrium phase behavior of the abovementioned oil, surfactant and/or cosolvent with aqueous media. The LFCS was first suggested by Pouton [15] for lipid formulations which once more modified and are currently being developed under the consortium based on their dispersion and the physicochemical characteristics of the formulation components. Previously Pouton and his group have studied the influence of mixed glycerides (blends of mono-, di-, and tri-glycerides) on the phase behavior of ternary glyceride/surfactant/water systems, but only to a minor extent. As glycerides (alone or blended) are typically good solvents of drugs and they promote absorption of water and self-emulsification, it was of particular interest of the present study to find the appropriate glyceride/surfactant blends which are able to solubilize a large proportion of water [16]. These studies were carried out to obtain a comprehensive understanding of how phase behavior varies for different lipid-surfactant compositions as they are diluted with water. In addition to these investigations, the particle size distribution of the formulations after dispersion in water and the appearance of the emulsion droplets were analyzed as a function of different lipid composition. As lipid systems vary in the efficiency of self-emulsification, the particle size distribution was particularly necessary to evaluate the effect of composition on the emulsification properties and the influence of choice of oil and surfactant on drug incorporation. 531
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
identified by making up 30-40 mixtures in the appropriate area of the phase diagram. Once an approximate map was available additional samples were prepared to determine boundary regions more precisely. Other phase boundaries of the phase diagram were also determined using the similar approach however, for practical reasons, given the number of phase diagrams investigated, in some cases the position of phase structures (lamellar/hexagonal etc.) was not determined. Phase behavior was examined using samples held at equilibrium for 48 h at 20 and 37 °C with the aid of a water bath (Ratek Instruments Pty. Ltd., Boronia, Victoria). Phase behavior was assessed initially by visual observation, classifying mixtures as one-phase or multiphasic (turbid) mixtures. LC phases were identified using an Allen viewer fitted with cross-polarizing filters, which were further identified as hexagonal or lamellar, whenever possible, based on the birefringence patterns observed under crossed polarized light of an optical microscope (Carl Zeiss, Axiolab, Jena, Germany) employing a magnification of 10-40× [18, 19]. To ensure uniformity, during the making each mixture was thoroughly blended with a Vm1 vortex mixture (Ratek Instruments Pty. Ltd., Victoria), and heated if necessary. Sample mixtures were weighed into and stored in glass tubes with water-tight closures (13×100 mm Pyrex, lined screw cap, Corning, United States).
I. Materials and Methods 1. Materials
Miglyol 812 (M812, medium chain triglyceride, C8-C10), Imwitor 988 (I988, medium chain mono- and diglycerides), Imwitor 308 (I308, 98 % medium chain monocaprylate) and propylene glycol (PG, 98 % pure) were supplied by Sasol Germany GmbH, Werk Witten, Germany. Capmul-PG8 (C-PG8, medium chain monocaprylate) was supplied by Abitec Corporation, Lloyd Hause, Janesville, WI, United States. Soybean oil (long chain triglycerides, 99 % pure) was obtained from Sigma-Aldrich Co, St. Louis, MO, United States. Maisine 35-1 (Maisine, long chain glyceryl monolinoleate) was provided by Gattefossé, Saint-Priest, France. The non-ionic surfactants used were polyoxyethelene-(20)-sorbitan monooleate (Tween 80, HLB-15) and polyoxyethelene-(20)-sorbitan trioleate (Tween 85, HLB-11), supplied by Sigma Aldrich Pty. Ltd., Castle Hill, New South Wales, Australia. 1 M HCl, which was diluted to obtain 0.1 M solution, was purchased from Merck (Darmstadt, Germany). All excipients were used as received without any further purification. Water used in this study was obtained from a Milli-Q water purification system (Sartorius, Göttingen, Germany).
2. Sample preparation and determination of ternary phase diagrams
3. Particle size determination
It was of particular interest to this study to determine the position of the phase boundary representing the maximum mass of water that can be solubilized by each glyceride/surfactant mixture. The phase boundaries of several phase diagrams were mapped out for the glyceride/surfactant and/or cosolvent with water, in some cases with phosphate buffer saline (PBS) and 0.1 M HCl (Table I) to find which combinations of mono-, di- and tri-glycerides, C-PG8 with Tween 80 (T80) or Tween 85 (T85) give rise to large regions of optically transparent mixtures (microemulsion regions) [16]. Samples for phase studies were prepared at three different stages such as primary mixtures, secondary mixtures and ternary mixtures at various weight ratios. Water was added progressively into each primary anhydrous glyceride-surfactant mixture. The transition from a clear to a cloudy solution as water was added indicated that the maximum solubilization had been exceeded [17]. For each phase study, a total of 80-100 mixtures were prepared to examine the whole phase diagram. Water uptake by glyceride-surfactant mixtures, to form water in oil (w/o) microemulsions produced transparent, apparently single phase regions, which were designated L2, using the usual terminology for the oil-rich phase. Initially the shape and size of the L2 region was
An understanding of the factors governing the particle size of resultant emulsions is important in designing any successful lipid formulations, particularly SEDDS/SMEDDS. In the current studies, emulsification performance of MCT and the blending of mono-, diand tri-glycerides were compared with long chain triglycerides (LCT) and similar product such as Maisine 35-1 (Maisine mono-glycerides: 32-52 %, di-glycerides: 40-55 %, and tri-glycerides: 5-20 %) [20] using surfactant T80. There are several techniques available at present to measure the particle size of dispersed systems. In this study, the Zetasizer 3000 (Malvern Instruments Limited, United Kingdom) was used by a combination of quasi-elastic laser scattering with photon correlation analysis to evaluate the particle size distribution (small droplets within 10-3000 nm) of the emulsions after dispersion in water The concentration used was selected to give a suitable obscuration value in the Zetasizer. A count rate of 50-500 Kcps was used to ensure accurate measurements (sample dilution was 1 in 1000, which did not alter the particle size). This analysis was performed on sample volumes of 1 mL using glass cuvettes. All measurements were performed at room temperature in triplicate, with 10 minute runs, using a 400 mm aperture. Prior to commencing the study, commercially available latex beads (Duke Scientific, Palo Alto, CA, United States) of known diameter (220 nm ± 6nm - NIST traceable, for reference standard) was subjected to size determination by the operating conditions of the instrument described above. Results were expressed as the Z-average mean which is the harmonic intensity-averaged particle diameter.
Table I - Components used to construct the equilibrium phase diagrams. Phase diagram
Figure
Oil
Surfactant
Aqueous phase
Medium chain triglycerides
1 2
M812 M812
T85 T80
Water Water
Medium chain glyceride blends
3 4 5 6 7
M812/I988 M812/I988 M812/I988 M812/I988 M812/I988
T85 T85 T80 T80 T80
Water Water Water Water Water
Medium chain mono- and diglycerides
8 9
I988 I988
T85 T80
Water Water
Medium chain mono-glycerides
10 11 12a 12b
C-PG8 I308 I308 I308
T80 T80 T80 T80
Water Water PBS 0.1M HCl
Cosolvent
II. Results 1. Correlation between self-emulsification and phase behavior
Using the equilibrium phase behavior studies, it is possible to obtain the mechanisms of self-emulsification process [21]. The phase regions in the current studies were named using conventional nomenclature system. In the ternary phase diagrams, represented in Figures 1-12, oil-continuous regions were denoted L2 (clear isotropic oily system), water continuous regions L1 (clear isotropic aqueous system) and the liquid crystalline phases were denoted LC. These LC structures were studied in detail (described further below) only for some of the ternary systems under optical microscope, which may contain either hexagonal or lamellar phases. For practical reasons no detailed attempt was made to quantify the volume fractions of the phases presented in the multiphasic mixtures.
PG
M812: Miglyol812, I988: Imwitor988, C-PG8: Capmul PG8, I308: Imwitor308, T85: Tween85, T80: Tween80, PG: propylene glycol and PBS: phosphate buffer saline. 532
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 1 - Triangular phase diagram of the M812/T85/water system (representing a typical LFCS Type II system) at (a) 20 and (b) 37 °C. L1 is water continuous phase, L2 is oil continuous phase, L1+L2 is two phase emulsion, L2+Lα is two phase oil and lamellar liquid crystals region, respectively. Identity and location of each phase is determined by polarized optical microscope. The dotted line denotes the area where lamellar phases were observed. Key: M812: Miglyol 812 and T85: Tween 85.
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
Figure 3 - Triangular phase diagram of the M812:I988 (7:3)/T85/water system (Type II) at (a) 20 and (b) 37 °C. The broken line drawn near the water corner at 37 °C shows the three phase emulsion (L1+L2+Lα). When the liquid crystalline phases are denoted as Lα, these mixtures contained lamellar phases. Key: M812: Miglyol 812, I988: Imwitor 988, and T85: Tween 85.
Figure 4 - Triangular phase diagram of the M812:I988 (3:7)/T85/water system (Type II) at (a) 20 and (b) 37 °C. (L2+LC) represents a two phase system of liquid crystal dispersed in oil phase, and (L1+L2+LC) emulsion with liquid crystalline phase. When the liquid crystalline phases are denoted as LC these mixtures may contain either hexagonal or lamellar phases. The solid line denotes the area where a lamellar phase is formed. Key: M812: Miglyol 812, I988: Imwitor 988, and T85: Tween 85.
Figure 2 - Triangular phase diagram representing the M812/T80/water system (LFCS Type IIIA) at (a) 20 and (b) 37 °C. Water continuous phase is designated L1, oil continuous phase is L2 and liquid crystal phase “white birefringence” is LC. Key: M812: Miglyol 812 and T80: Tween 80.
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J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 5 - Triangular phase diagram of M812:I988 (3:7)/T80/water system at (a) 20 and (b) 37 °C (Type IIIA systems). Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, two phase emulsion is (L1+L2) and liquid crystal phase “white birefringence” is LC, which contained lamellar structure. Key: M812: Miglyol 812, I988: Imwitor 988, and T80: Tween 80.
Figure 7 - Triangular phase diagram for the system M812:I988:PG (2:2:1)/T80/water system at (a) 20 and (b) 37 °C. Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, two phase emulsion is (L1+L2). Key: M812: Miglyol 812, I988: Imwitor 988, PG: Propylene glycol and T80: Tween 80.
Figure 6 - Triangular phase diagram of the M812:I988 (7:3)/T80/water system at (a) 20 and (b) 37 °C (Type IIIA systems). Oil continuous phase is designated L2, surfactant phase containing liquid crystal is (S+LC), and two phase emulsion is (L1+L2). Key: M812: Miglyol 812, I988: Imwitor 988, and T80: Tween 80.
Figure 8 - Triangular phase diagram of the I988/T85/water system at (a) 20 and (b) 37 °C. Different compositions of the formulation result in the formation of different phase structures. Oil continuous phase is designated L2, in presence of liquid crystal is L2+LC, two phase emulsion is (L1+L2). Key: I988: Imwitor 988, and T85: Tween 85.
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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
Figure 9 - Triangular phase diagram of the I988/T80/water at (a) 20 and (b) 37 °C (Type IIIA). Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, and two phase emulsion is (L1+L2). Key: I988: Imwitor 988, and T80: Tween 80.
Figure 11 - Triangular phase diagram of the system I308/T80/water at (a) 20 and (b) 37 °C (Type IIIB), adapted from ref. 23. Oil continuous phase is designated L2, two phase emulsion is (L1+L2) and surfactant phase S. Both lamellar and hexagonal phases are present in the multiphase region of the phase diagram. Key: I308: Imwitor 308, and T80: Tween 80.
Figure 10 - Triangular phase diagram of the system C-PG8/T80/ water at (a) 20 and (b) 37 °C (Type IIIB). Oil continuous phase is designated L2, in presence of liquid crystal is L2+LC, two phase emulsion is (L1+L2) and the two phase emulsion in presence of liquid crystal is (L1+L2+LC). Key: C-PG8: Capmul PG8 and T80: Tween 80.
Figure 12 - Triangular phase diagram of the I308/T80/water system at 37 °C. (a) PBS (pH-7.5) and (b) 0.1 M HCl (pH-1.1) were used for the aqueous component. Oil continuous phase is designated L2, two phase emulsion is (L1+L2) and the two phase emulsion in presence of liquid crystal is (L1+L2+LC). Key: I308: Imwitor 308, and T80: Tween 80.
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J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
For example (L1+L2) represents a turbid emulsion system in the phase diagram but the volumes and precise compositions of each phase were not investigated in detail. Typically (L1+L2) mixtures were formed close to the oil-water axis. Multiphasic dispersions of L1, L2 and LC phases also produced emulsions that had the same outward appearance as L1+L2 emulsion. It was possible to distinguish L1+L2+LC mixtures from L1+L2 when the Allen viewer revealed that LC was also present in the turbid mixtures. A clear strong birefringent LC observed adjacent to L2 regions was identified as L2+LC mixtures. L1+LC mixtures were mostly identified near the water-rich corner. In some of the phase diagrams an isotropic viscous gel-like phase was identified as Surfactant phase (S). This was considered to be a single phase rich in surfactant but with large amounts of water solubilized in it. These mixtures were difficult to distinguish from the L2 phase except looking at the viscosity.
Further dilution of this system with water produced liquid crystalline phases in combination with L2 or L1 and L2. The L2 region included a finger-like projection towards the water apex at 60-70 % surfactant (Figure 2b). This is a typical feature of SEDDS phase diagrams which is thought to correspond with rapid emulsification. 3.2. Medium chain glyceride blend Figure 3 depicts the phase diagram for M812:I988 (7:3)/T85/water system, where the oil component was a blend of M812 and I988 in the weight ratio 7:3. The anhydrous system solubilized a considerable mass of water, compared to the corresponding M812 system, as indicated by the extensive L2 region. Solubilization of water was pronounced at T85 concentrations between 40-50 %, up to 40 % water by weight at the maximum, and featuring the characteristic finger-like projection of the L2 region towards the water axis. The blending of M812/I988 at 7:3 ratios improved the performance of the LFCS Type II system compared to the previous system that contained M812 alone. This study suggests the substantial differences in the phase diagrams when the polarity of the oil phase is increased by the blends of mono-, di- and tri-glycerides with T85. It is important to note that M812 itself and the mixture of M812/I988 (7/3, w/w) are very poorly soluble in water. Figure 4 also shows the similar Type II systems with a different oil ratios of M812/I988 (3/7, w/w). The boundary of the L2 region of this phase diagram was slightly reduced in area in comparison with that shown in Figure 3. The large mass of water (approx. 35 %, Figure 4a) was taken up by the oil/surfactant (50/50, % w/w) anhydrous formulation. A slender (L1+L2+LC) phase at 20 °C and (L1+LC) phase at 37 °C (Figure 4b) was observed close to the water axis. Given the benefit gained by increasing the polarity of the oil phase with T85, it was appropriate to examine what effect the oil polarity had on LFCS Type III systems formed with hydrophilic surfactant T80. The replacement of T85 with T80 produced a significant change in the shape and extent of the L2 region, reflecting the affinity of T80 for water. These systems were Type IIIA systems, capable of producing very fine dispersions (< 100 nm, optically clear if higher concentration of T80 used). Mixtures of T80 with M812/I988 (3/7) were very good self-emulsifying systems which solubilized a large mass of water (Figure 5a-b). A transparent surfactant phase (S) was identified next to L2 region along the surfactant/water axis. This high viscous S phase contained hexagonal structure between 40 % & 50 % water. There was a bluish (transition between transparent and emulsion) phase observed at 37 °C towards the water apex within L2+Lα phase region (Figure 5b). The phase diagram for M812:I988 (7:3)/T80/water system also represents LFCS Type IIIA shown in Figure 6. This phase diagram can be compared with M812:I988 (7:3)/T85/water of Type II system (Figure 3) to see the effect of surfactant on the phase behavior. In fact, there was a significant difference to the water uptake towards the surfactant apex of the phase diagram (Figure 6a-b). At 37 °C, when the anhydrous mixture contained 70-80 % T80, the system was capable of absorbing an equal mass of water before a phase change occurred. With addition of more water, the two phase region S+LC (surfactant in equilibrium with liquid crystals) was formed in this system. Including PG in the oil phase (blended with M812 and I988) was also explored as a single glyceride component to increase the polarity of M812:I988:PG (2:2:1)/T80/water system (Figure 7). This system produced a large isotropic L2 phase region, which was reversed to L1 towards the water corner. Large (L1+L2) emulsion region was present along oil-water axis with addition of 20-30 % T80. However, above 30 % T80 there were large areas of mutual solubilization of the excipients and water. At extremely high surfactant concentration (9099 % T80) within L2 region, S phase was extended half-way towards the water axis. Another small (L1+L2+LC) region was present close to the water apex which was evident at 20 °C (Figure 7a) but disappeared to L1+L2 (emulsion) region at 37 °C (Figure 7b).
2. Characterization of maximum water uptake (L2/L1 phase)
The ternary phase diagrams constructed in the study indicate the phase changes which occurred when anhydrous oil-surfactant mixtures are mixed with water. Many of the systems were capable of absorbing considerable amounts of water, so that an extensive L2 region containing up to 55-60 % water. Figure 11 shows a typical example of the L2 phase that formed on dilution of a LFCS Type IIIB formulation. This shows the phase diagram of a simple but effective formulation comprising I308 and T80, which allowed the system to solubilize a large mass of water. However in this system, the L2 phase transformed into a transparent aqueous microemulsion or micellar system (L2 to L1, i.e., w/o to o/w microemulsions) [22]. These phases illustrate differences which are expected to influence the mechanism and efficiency of self-dispersion, which was only possible by determining the phase diagrams.
3. Introduction to the phase diagrams
The phase diagrams described in the studies depicted in Figures 1-12 were constructed at ambient (20 °C), and body (37 °C) temperature to investigate whether there were any substantial differences in phase behavior over the range ambient to body temperature. 3.1. Medium chain tri-glyceride The triangular equilibrium phase diagram for the M812/T85/water system at 20 °C and 37 °C is shown in Figure 1. Among the various phases formed by these three components, a clear and transparent oily region (dark grey L2 area) has been identified close along to the oilsurfactant side. This system represents an example of LFCS formulation Type II. Combinations of M812 and T85 containing between 30 % and 60 % produced SEDDS that have no water-soluble components. Figure 1b indicates that a maximum of 15 % water was solubilized by the anhydrous mixtures, i.e. at higher surfactant concentrations. On further dilution of the anhydrous systems with water, a phase change resulted in a distinct two phase region (L2+LC), which is typical for Type II systems (LC containing lamellar phase, Lα). The emulsification with non-ionic surfactant T85 in the system is thought to involve dynamic formation of lamellar liquid crystals at the oil-water interface. This would allow penetration of water into the bulk material by way of aqueous channels, causing an increase in interfacial instability, resulting in droplets being propelled from the inter phase. However the M812/T85 system is not as rapidly dispersing as related systems which include mono- and diglycerides. Figure 2 shows the ternary phase diagram for the M812/T80/ water system. The L2 region had a modest area, which indicated that relatively little water was solubilized by this system. In the L2 region, a maximum of 15 % water was solubilized at 20 °C when the weight proportions of oil and surfactant were equal. Solubilization of water was significantly increased to 28 % at 37 °C, which may indicate that emulsification is more efficient at 37 °C (Figure 2b). 536
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
3.3. Medium chain mono- and di-glyceride blend The phase diagram of the I988/T85/water system (Figure 8) provides information regarding the effect of mono- and di-glyceride on the phase behavior. Solubilization of water within the L2 region is moderate at most oil-surfactant anhydrous mixtures. Just the inclusion of MCT could have significant phase changes as evident by M812:I988/ T85 system (Figure 3). In the system with only I988 (Figure 8), the pointed finger-like region formed at approximately 50 % w/w glyceride/surfactant ratio. It is worth knowing that I988 is a polar lipid which was able to solubilize 15 % water itself in the absence of T85 (Figure 8a). Replacing T85 with T80 in the system resulted in more isotropic regions in the phase diagram shown in Figure 9. The L2 and S phases were clearly evident with reverse L1 phase. A finger-like projection in the L2 area was centered at the 50/50 ( %w/w) glyceride/surfactant ratio.
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
on the phase behavior was determined by comparison of the relevant phase boundaries with those recorded on the corresponding diagrams constructed with water (Figure 11). There were some differences between the phase diagrams (Figure 11b versus Figure 12a-b) but in general the features recorded with water with preserved. In the alkaline buffer system, liquid crystalline (LC) phase was not existent with oil (L2) and water (L1) continuous phases at 37 °C (Figure 12a). Below the L2 region in the I308/T80/PBS system, the (L1+L2) emulsion was formed. In contrast LC was still present within the L1+L2 phase in I308/T80/0.1 M HCl system at 37 °C (Figure 11b). The location of the L2 phase regions in both systems were not significantly affected by the variation of pH. These systems will need to be investigated further to determine whether dilution in acid or buffered systems can have any effect on the solubilization and dispersion of the drug.
5. Effect of temperature on the phase diagrams
Ternary phase diagrams at two different temperatures (20 and 37 °C) were constructed only to determine the possible effects of temperature on the dispersion process. It was also of interest to compare the areas of various phase regions and the effects of temperature on liquid crystal formation. A size reduction of L2 phase with increase of the temperature was apparent in the phase diagram of M812:I988 (7:3)/T85/water system (Figure 3). Temperature increases also commonly decreased the area of regions which included liquid crystal. For example, the I988/T80/ water system (Figure 9b) had a reduced L2+LC phase region at 37 °C. The L2 phase region in the I308/T80/water system (Figure 11) was affected significantly by temperature variation. A temperature increase to 37 °C eradicated the wax formation in the oil (I308) corner due to melting of the mono-glyceride content (Figure 11b). On the other hand, the area of L1 phase region was increased at 37 °C. This appeared to be due to a reduction in the viscosity of the S phase which resulted in the assignment of a larger L1 phase region. In common with other systems, the LC phases melted as temperature was raised so that the three phase region (L1+L2+LC) disappeared at 37 °C and became L1+L2. I308 (waxy mono-glycerides) was able to solubilize almost 10 % more water at 37 °C than at ambient temperature which indicates that self-emulsification is likely to be more efficient at body temperature.
3.4. Medium chain mono-glyceride C-PG 8/T80/water represents the LFCS Type IIIB system, which shows the effect of using only mono-glyceride with hydrophilic surfactant (Figure 10). This composition, which is highly water-soluble in nature, produced a different shape of L2 phase, presented as hooklike dark grey area. With C-PG8, at high concentrations of surfactant (above 70 %), there was substantial solubilization of water, particularly at 37 °C (Figure 10b), although the uptake of water at lower surfactant concentrations was not as significant as for other Type III formulation systems (Figure 5b). Replacement of the C-PG8 with I308 in the glyceride (oil) corner created a remarkable change in the extent of water absorption by the L2 phase (Figure 11). The essential difference between C-PG8 and I308 is one extra hydroxyl group per molecule in I308. A wide range of clear transparent mixtures covering a surprising variety of mixture compositions was evident in Figure 11b. In this regard, I308 is a rather unique excipient which may be very useful for lipid formulations. In addition, the isotropic L2 region was extended up to S and L1 phases. A bluish transparent phase was observed within L2 phase at very high water contents in regions that were thought to be colloidal in nature (Figure 11b) [23]. At concentrations of T80 below 50 %, a phase change occurred when excess water was added, producing L2+LC systems (Figure 11a). The 50 % T80 system dispersed to produce a fine emulsion characteristic of SMEDDS, though it should be noted that this precise formulation has not been investigated by other authors, and it may have unique properties being so rich in mono-glycerides. Although I308 was a useful excipient which promoted absorption of water and efficient emulsification, it had the disadvantage that the pure mono-glyceride was semi solid waxy at ambient temperatures. For this reason, the phase diagram of the I308/T80/water system was determined in detail at 20 °C in the vicinity of the I308 apex to identify practical problems that might occur due to crystallization of waxy mono-glycerides. The region in which crystals were detected is displayed as the dark gray area in Figure 11a. This data was obtained by keeping the samples at 20 °C for 15 days before making an assessment by visual inspection. The waxy phase melted between 20 and 37 °C. This data indicated that the use of I308 as the single oily component may not be practical at I308 concentrations above 40 % because the anhydrous materials may become segregated if crystallization of mono-glyceride occurs on storage.
6. Investigation of liquid crystalline structures using optical polarizing microscopy
The presence of liquid crystalline material in some of the formulations was initially identified in the vials with the samples viewed between crossed polarizers (Allen viewer) to probe the structure of the LC phases. The hydrated samples with different water content were then examined under a polarizing light microscope (samples placed between micro slides and cover slips) to detect the presence of optically anisotropic phases (lamellar and hexagonal phases). The textures of the hexagonal and lamellar liquid crystalline materials when viewed by polarizing microscopy are distinctly different. In the phase studies, a large anisotropic area containing lamellar (Lα) phase (Figure 13a-b) exists in many of the ternary glyceride/ surfactant/water systems (Figures 1, 3 and 11) at water contents ranging from 15 % to as much as 90 %, and these phases are perhaps more prevalent at 20 °C. The Lα phase consists of bilayers separated by water, forming the comparatively low viscosity Lα liquid crystalline phase, the viscosity being another indicator of structure. In M812:I988 (3:7)/T80/water system (Figure 5), hexagonal liquid crystal (H1) and Lα liquid crystal phases were successively formed within Surfactant (S) region. It was thought that cylindrical micelle structures were closely packed in a hexagonal array in the H1 phase which could be identified by its optical microscopy pattern (Figure 13c-d). The hair-like texture of hexagonal phase (H1) was seen at 40 % to
4. Influence of pH on the phase behavior
Dispersion of lipid-based formulations in vivo takes place either in the gastric or intestinal fluids, rather than in pure water. In order to investigate whether the pH of the aqueous medium influences the phase behavior of the I308/T80 formulation, phase diagrams were investigated using phosphate-buffered saline (PBS, pH-7.5, Figure 12a) and 0.1 M hydrochloric acid (pH-1.1, Figure 12b) at 37 °C. The effect of pH 537
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 14 - Emulsion droplet diameter of dispersions formed by selfemulsification systems with M812/I988 (7/3, % w/w), SOB/I988 (7/3, % w/w), M812/Maisine (7/3, % w/w) and SOB/Maisine (7/3, % w/w) each in combination with Tween 80, as a function of surfactant concentration. Formulations were emulsified with water at room temperature for 10 min and then analyzed by photon correlation spectrometer (PCS). Data are mean ± SD (n = 3). Key: M812: Miglyol 812, I988: Imwitor 988, and SOB: soybean oil.
Figure 13 - Representative photomicrographs of (a) & (b) lamellar liquid crystalline phase structures, and (c) & (d) hexagonal liquid crystalline structures. These images were taken using samples of I308/Water (60/40, % w/w) and T80/Water (50/50, % w/w) between cross polarizers under light optical microscope. Key: I308: Imwitor 308, and T80: Tween 80.
low concentrations the emulsions were coarse at surfactant concentrations lower than 40 % w/w. A few studies have reported similar trends in droplet size with increase in surfactant concentration for various self-emulsifying systems [25]. Small particle size did not necessarily correlate with rapid emulsification. At high surfactant contents > 7080 %, the dispersions formed were essentially transparent but these dispersions often took much longer to reach completion. Various compositions of M812:I988 (7:3) and T80 appeared to emulsify quite rapidly and produced fine dispersions at room temperature. The most efficiently dispersing systems (dispersed ≤ 1 min in water) were formed at 30 to 50 % surfactant concentrations, producing dispersions with particle sizes less than 300 nm. M812:I988 (7:3)/T80 systems containing above 90 % T80 took > 5 min to disperse. The replacement of MCT with LCT (SBO) to produce the systems affected considerably the efficiency of emulsification. Generally mixtures containing SBO produced more turbid, coarser dispersions. When these systems were analyzed using the Malvern Zetasizer the apparent mean particle size was relatively high, close to or in excess of 1 μm. The apparent particle size distributions and dispersion times of M812:Maisine (7:3)/T80 and SBO:Maisine (7:3)/T80 formulations were found to be similar, producing sub-micron dispersions at surfactant concentrations above 30 %. The M812:Maisine system produced significantly finer emulsions but a limited particle size differences between the two systems were not likely to determine the differences between their performances. Thus in the long chain mixed glycerides there appeared to be little advantage gained by including MCTs.
50 % water in surfactant-water binary mixtures (T80/water composition in Figure 11). When 5 % I308 was added to the binary surfactant-water system, the H1 phase reverted to a multiphasic region (containing Lα) as shown in Figure 11a. An optically anisotropic liquid crystalline region in binary mixtures of I308 with 30-60 % (w/w) water formed lamellar structures (Figures 11a and 13a). An increase in temperature (to 37 °C) strongly reduced the presence of the Lα phase and transforms it into a fluid isotropic L2 phase. However, I308 did not appear to exhibit the classical phase sequence: L2 (reversed micellar), HII (reversed hexagonal), QII (reversed cubic), and Lα (lamellar). This sequence is often observed upon hydration of poorly soluble amphiphilic molecules [24].
7. Effect of formulation on the efficiency of self-emulsification
A series of mixtures comprising medium chain glycerides and long chain glycerides with hydrophilic surfactant T80 were prepared and their self-emulsifying properties were studied using visual examination and particle size analysis. T80 is a popular choice of surfactant for formulation of SEDDS/SMEDDS, so it was of interest to compare the efficiency of the systems formulated with T80. Initially, anhydrous glycerides and surfactants were mixed in different proportions (10-100 % w/w) at room temperature so that a range of surfactant concentration could be investigated. One hundred milligrams formulation was introduced into 10 mL (1 in 100 dilution factor) of water in a glass beaker at room temperature, and the contents were agitated gently with a glass rod. The tendency to emulsify spontaneously and also the progress of emulsion droplets were observed against time. In this way, it was easy to distinguish between formulations that formed microemulsions, which were clear or slightly blue translucent dispersions, and formulations that formed crude emulsions on dispersion. An estimation of the time required for complete dispersion in water at ambient temperature was noted in triplicate. According to the visual inspection, < 5 s represented spontaneous dispersion whereas > 1 and > 5 min indicated the slow and longer dispersion times for the samples, respectively. Figure 14 illustrates the influence of the chain length (MCT vs LCT) over a wide range of surfactant concentration. For M812/I988 (7/3) and SBO (Soybean oil)/I988 (7/3) w/w, it is evident that the size of the emulsion droplets decreased with increase in the concentration of T80, but in a more marked manner for the long chain glycerides. At
III. Discussion
A critical characteristic of a successful lipid formulation is its ability to emulsify rapidly and disperse well in the aqueous media. Currently, many pharmaceutical excipients are not miscible themselves and poorly dispersible as they are diluted in aqueous media, which is creating a real challenge in terms of developing formulation. The ternary phase diagrams constructed using three component systems in the current research were adequate in determining phase transitions and developing optimal lipid formulations. However, in some cases, such as the identification of the phase structures of lamellar and hexagonal phases, a great deal more work would be required to pin-point the exact phase boundaries. These phase changes between different liquid crystalline phases could be identified by optical microscope, but were squeezed into a relatively small area (Figures 1-12). It is well known that self-emulsification is specific to the characteristics of the oil/surfactant affinity, surfactant concentration and the 538
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
temperature at which self-emulsification occurs [7]. From the results of the current studies it was satisfying to observe that the inclusion of mono- and di-glycerides in the formulation systems led to increased efficiency of emulsification. However, to select any successful formulation, understanding of the behavior of mono-, di- and tri-glycerides and either hydrophilic or lipophilic surfactant when blended together is considered to be an initial step. Equilibrium phase studies can give a clear picture of how the excipients mix remains miscible, by identifying mixtures which can tolerate small changes in water content, such as might arise during encapsulation or drying of the manufactured product, as the capsule reaches its final hydration state. In the current studies, MCT oil with mixed mono- and di-glycerides were predominantly used for the design and the assessment of selfemulsifying formulations. T80 (HLB=15) and T85 (HLB=11) were chosen deliberately as a pair of chemically related materials, which represent a water-soluble and water-insoluble surfactant, respectively. They are known to have low toxicity, at least in the case of T80, and have been used popularly in many pharmaceutical products. The inclusion of mixed glycerides improved the quality of the emulsions reported in previous studies. It was reported that a selfemulsifying formulation of a lipophilic drug WIN 54954 with a combination of MCT (Neobee M5)/ non-ionic surfactant (Tagat TO)/ drug (40/25/35, % w/w), emulsified rapidly upon gentle agitation at 37 °C producing emulsions with mean droplet diameter of less than 3 µm [21]. In the current studies, it is also observed that mixed mono-, di- and tri-glycerides with surfactants have a higher tendency to uptake large amount of water and form emulsions of small particle size. The blending of tri-glycerides with mixed mono-and di-glycerides had a beneficial effect of improving water solubilization. In addition, the inclusion of PG in mixed glycerides produced systems with extensive amounts of water solubilization (Figure 7). Extensive water absorption is generally associated with rapid emulsification and fine particle size [2]. Phase diagrams of T80 with I308 in particular were found to have very large regions which are optically transparent (Figure 11). This transparent region could include L2 (oil-continuous) and L1 (water-continuous) phases which are microemulsions containing a considerable variety of microstructures [22, 26]. Many of these systems were also rich in surfactant, and could be referred to as “surfactant phase-S” with large masses of co-solubilized water and oil. The I308/ T80 system is a highly promising formulation since a large area of the phase diagram is covered by well emulsified systems. The work in this research has also shown that the emulsification properties of mono-glyceride/di-glyceride/tri-glyceride/Tween-based self-emulsifying systems are greatly dependent on the composition, as more hydrophilic mixtures showed a higher tendency to solubilize water and form emulsions of small particle size. It was evident that at very high concentration of surfactant (70-90 % w/w), microemulsions were formed with most of the glycerides (oils). These systems tended to be denser than water and dispersed slowly after the bulk material had sedimented. At lower concentrations of surfactant (20-60 % w/w) the dispersions were turbid, although they often self-emulsified in an efficient manner. In addition, these systems were found to be temperature sensitive in the temperature range 20~37 °C. Like most changes observed with temperature in the phase diagrams, this was likely to be due to the progressive reduction in solvation (i.e. hydration) of the surfactant ethoxylate chains which occurred as the temperature was elevated. Within the scope of the present study, LFCS Type II formulation systems were modeled by blends of glycerides and T85. Blends of M812 and T85 self-emulsified within a minute but were not a good system to solubilize much water (L2 phase region). However, the solubilization was improved significantly by blending of tri-glycerides with mixed mono- and di-glycerides. This is in agreement with similar unpublished work by Pouton and colleagues using the surfactant Tagat TO, which is also an ethoxylated trioleate ester with an HLB
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
close to that of T85. Type IIIA formulations were formed by replacing T85 with T80. They had improved emulsification properties and comparably reduced particle size. In these systems the medium chain mono-, di- and tri-glycerides could be replaced by long chain mono-, di- and tri-glycerides but with increase in particle size. The rate of emulsification and the particle size of the dispersed formulation are the key descriptors of the efficiency of self-emulsification process, though according to the most studies it was considered that a visual inspection of rate of emulsification was sufficient to compare formulations. I308/ T80 system could be considered to be a Type IIIB SMEDDS, but it may represent a new class of Type IIIB formulation which can have a high content of medium chain mono-glyceride, for example the system 50 % I308/50 % T80. Generally, rapidly emulsifying Type IIIA & IIIB formulations were efficient self-emulsifying systems with regard to particle size, and resulted in emulsions in the submicron range on aqueous dispersion. Some classes of lipids were able to form viscous liquid crystalline structures (lamellar/hexagonal) in excess water (Figure 13). The ability of alkyl glycerates to form reverse hexagonal phases was recently reported by Boyd et al. [27] There has been increasing interest in the potential pharmaceutical utility of liquid crystalline structures formed by polar lipids and or surfactants in excess water [28, 29]. The viscous hexagonal liquid crystalline phases offer considerable scope for drug delivery applications. They are of particular interest, as their structures provide a diffusion barrier to drug release when the drug is incorporated into the matrix on formation; thus they have potential as sustained-release matrices [30]. The effect of liquid crystalline structure on drug absorption after oral administration of liquid crystal-forming lipids is not yet clear. * Overall the current studies confirmed that mixed glycerides are good self-emulsifying systems, which can promote absorption of water and rapid dispersion. As they are good solvents for drugs, their attributes can be utilized advantageously to fit drug delivery challenges (particularly water insoluble drug), making lipid-based drug delivery systems a successful approach. Design rules for the choice of oil, surfactant, as well as the surfactant ratio for maximum solubilization may be established by the construction of phase diagrams. Amongst the LFCS systems the most efficient formulations were produced by ethoxylated sorbitan ester in combination with I308, which were categorized as Type IIIB. It was also possible to formulate good SEDDS with T80 as long as I988 or Maisine 35-1 were included.
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Constantinides P.P. - Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. - Pharm. Res., 12, 1561-72, 1995. Pouton C.W. - Lipid formulations for oral administration of drugs: non-emulsifying, self-emulsifying and 'self-microemulsifying' drug delivery systems. - Eur. J. Pharm. Sci., 11, Suppl. 2, S9398, 2000. Gershanik T., Benita S. - Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs. - Eur. J. Pharm. Biopharm., 50, 179-88, 2000. Pouton C.W. - Formulation of self-emulsifying drug delivery systems. - Adv. Drug Del. Rev., 25, 47-58, 1997. Pouton C.W. - Self-emulsifying systems for drug delivery. University of London, 1982. Wakerley M.G., Pouton C.W., Meakin B.J., Morton F.S. - Self emulsification of vegetable oil-non-ionic surfactant mixture: a proposed mechanism of action. - Am. Chem. Soc. Symp., Ser. 311, 242-255, 1986. Wakerley M.G., Pouton C.W., Meakin B.J., Morton F.S. - The
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effect of surfactant HLB on the self-emulsifying efficiency of nonionic surfactant-vegetable oil mixtures. - J. Pharm. Pharmacol., 38, 2, 1986. Capek I. - Degradation of kinetically-stable o/w emulsions. - Adv. Colloid Interface Sci., 107, 125-55, 2004. El-Mahrab-Robert M. et al. - Assessment of oil polarity: comparison of evaluation methods. - Int. J. Pharm., 348, 89-94, 2008. Wennerstrom H., Balogh J., Olsson U. - Interfacial tensions in microemulsions. - Colloids and Surfaces A, 291, 69-77, 2006. Lawrence M.J., Rees, G.D. - Microemulsion-based media as novel drug delivery systems. - Adv. Drug Del. Rev., 45, 89-121, 2000. Djekic L., Primorac, M. - The influence of cosurfactants and oils on the formation of pharmaceutical microemulsions based on PEG-8 caprylic/capric glycerides. - Int. J. Pharm., 352 (1-2), 231-239, 2007. Craig D.Q.M., Barker S.A., Banning D., Booth S.W. - An investigation into the mechanisms of self-emulsification using particlesize analysis and low-frequency dielectric-spectroscopy. - Int. J. Pharm., 114, 103-110, 1995. Subramanian N. et al. - Formulation and physicochemical characterization of microemulsion system using isopropyl myristate, medium-chain glyceride, polysorbate 80 and water. - Chem. Pharm. Bull. (Tokyo), 53, 1530-1535, 2005. Pouton C.W. - Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system. - Eur. J. Pharm. Sci., 29, 278-87, 2006. Hatton J. et al. - Safety and efficacy of a lipid emulsion containing medium-chain triglycerides. - Clin. Pharm., 366-371, 1990. Merdas A., Gindre M., Ober R., Nocot C., Urbach W., Waks M. - Water confined in reverse micelles: acoustic and densimetric studies. - J. Phys. Chem. B, 101, 10751-10756, 1997. Pouton C.W. - Self-emulsifying drug delivery systems, Assessment of the efficiency of emulsification. - Int. J. Pharm., 27, 335-348, 1985. Kossena G.A., Charman W.N., Boyd B.J., Dunstan D.E., Porter C.J. - Probing drug solubilization patterns in the gastrointestinal tract after administration of lipid-based delivery systems: a phase diagram approach. - J. Pharm. Sci., 93, 332-348, 2004. Gonikberg E. - Excipients: monograph content. - Pharmacopoeial Forum, 30 (3), 1975.
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Charman S.A. et al. - Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound. - Pharm. Res., 9, 87-93, 1992. Mohsin K., Long M.A., Pouton C.W. - Design of lipid-based formulations for oral administration of poorly water-soluble drugs: Precipitation of drug after dispersion of formulations in aqueous solution. - J. Pharm. Sci., 98, 3582-3595, 2009. Mohsin K., Alanazi F. - The fate of paclitaxel during in vitro dispersion testing of different lipid-based formulations. - J. Drug. Del. Sci. Tech., 22 (2), 197-204, 2012. Barauskas J., Landh T. - Phase behaviour of the phytantriol/ water system. - Langmuir, 19, 9562-9565, 2003. Devani M., Ashford M., Craig D.Q.M. - The emulsification and solubilisation properties of polyglycolysed oils in self-emulsifying formulations. - J. Pharm. Pharmacol., 56, 307-316, 2004. Sakya P., Seddon J.M., Templer R.H., Mirkin R.J., Tiddy G.J.T. - Micellar cubic phases and their structural relationships: the non-ionic surfactant system C12E12/water. - Langmuir, 13, 3706-3714, 1997. Boyd B.J. et al. - Lyotropic liquid crystalline phases formed from glycerate surfactants as sustained release drug delivery systems. - Int. J. Pharm., 309, 218-226, 2006. Shah J.C., Sadhale Y., Chilukuri D.M. - Cubic phase gels as drug delivery systems. - Adv. Drug Del. Rev., 47, 229-250, 2001. Drummond C.J., Fong C. - Surfactant self-assembly objects as novel drug delivery vehicles. - Curr. Opin. Colloid Interface Sci., 4, 449-456, 1999. Boyd B.J. et al. - A lipid-based liquid crystalline matrix that provides sustained release and enhanced oral bioavailability for a model poorly water soluble drug in rats. - Int. J. Pharm., 340, 52-60, 2007.
Acknowledgements The author is thankful to Dr. Michelle Long for her assistance during this study.
Manuscript Received 1 February 2012, accepted for publication 3 April 2012.
540
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery systems K. Mohsin1, 2*, C.W. Pouton1 1 Monash Institute of Pharmaceutical Sciences, Monash University, Australia Present address: Kayyali Chair for Pharmaceutical Industries, KSU, Riyadh, Saudi Arabia *Correspondence: [email protected]
2
The study was designed to investigate phase behavior during dispersion of anhydrous lipid formulations, and to investigate the effect of lipid/ surfactant ratios, and the presence of cosolvent on the performance of formulations. Equilibrium phase studies were extensively conducted using three component systems to investigate the phase changes of the anhydrous formulation in response to aqueous dilution. The influence of chain length and combination of mono-, di- and tri-glycerides on the phase behavior was studied. Droplet size studies were carried out to assess the influence of lipid and surfactant on the resultant droplet size upon aqueous dilution. The study shows that mixture of mono-, di- and tri-glycerides with equal amount of surfactant can promote great absorption of water and produce efficient self-emulsification systems (particularly, self-emulsifying/ microemulsifying drug delivery systems). Increasing the monoglyceride concentration within the oil component enhanced water solubilization significantly. Among the phase diagram studies, Imwitor 308/Tween 80 systems produced a large optically transparent nanodispersing region. The selection of glycerides seems to be the most vital oil component in designing optimal self-emulsifying lipid formulations. Key words: Lipid-based formulation – Self-emulsifying systems – Ternary phase diagram – Particle size analysis. From the last decade, there has been increasing focus on the utility of self-emulsifying/microemulsifying drug delivery systems (SEDDS/ SMEDDS) to enhance the oral bioavailability of large number lipophilic drugs [1, 2]. It has been well documented that SEDDS/ SMEDDS can be formulated with various lipid excipients, which are homogenous mixtures of natural or synthetic oils, water-soluble or insoluble surfactants or alternatively, one or more hydrophilic solvents and cosolvents [3]. The materials (i.e. vegetable oil derivatives, nonionic surfactants etc.) used in the systems are suitable for oral ingestion [4-6]. SEDDS/SMEDDS formulations are either water free or low in water content which allows encapsulation of a final unit dose in soft or hard gelatin. Thus they help to minimize irritation caused by the regular solid bulk drug substances with the gut wall. A number of early studies have shown that [7] only very specific combination of pharmaceutical excipients led to efficient self-emulsifying systems, which expected to self-emulsify rapidly in the aqueous contents of the stomach. As a result, it presents the drug in fine droplets of oil (< 5 µm) without any precipitation. Fine oil droplets should empty rapidly from the stomach and promote faster and immediate drug release throughout the GI tract. The principal characteristic of these systems is their ability to form fine oil in water (o/w) emulsions or mostly a single phase microemulsions (oil or water continuous phase) upon mild agitation when exposed to aqueous phases. It has a particular relevance in the understanding and the control of the formulation and stability of preparations in which water and oil coexist, such as dispersed systems [8, 9]. It is well known that aqueous mass transfers are likely to occur between the dispersed drops, through the oil continuous phase. These transfers are probably highly influenced by surfactants, which are necessarily present in the emulsions, and that give sometimes direct or reverse micelles, inside which the aqueous or oily transport will be made easier. Indeed, SMEDDS in another term “microemulsions” are thermodynamically stable, macroscopically isotropic and nanostructured mixtures, which often require high surfactant concentrations in order to microemulsify entire oil and water phase [10]. Single phase microemulsions are currently of interest to the pharmaceutical scientist as potential drug delivery vehicles due to their long term stability, ease of preparation, and considerable capacity for solubilization of a variety of drug molecules [11, 12].
It is worth mentioning that if a hydrophobic drug has adequate solubility in oil or oil/surfactant blends then SEDDS/SMEDDS can be a great option for their oral delivery [13]. According to many reports, medium chain fatty acids, mono-/di-, and tri-glycerides, particularly caprylic/capric mono-/di-glycerides have been used independently in mixed micelle emulsion, microemulsion and SEDDS/SMEDDS formulations and as absorption enhancers for a number of drugs, since they have been found to be physiologically well tolerated [14]. It is important for the current studies to note that glycerides are the oil component of the formulations. Within the scope of the present studies, it was required majorly to identify the self-emulsifying formulations using wide ranges of oils composed of medium chain mono-, di-, and tri-glycerides, water-soluble cosolvents and commonly used non-ionic surfactants following the lipid formulation classification system (LFCS) [15]. To fulfill this requirement, a series of phase diagrams were constructed to study the equilibrium phase behavior of the abovementioned oil, surfactant and/or cosolvent with aqueous media. The LFCS was first suggested by Pouton [15] for lipid formulations which once more modified and are currently being developed under the consortium based on their dispersion and the physicochemical characteristics of the formulation components. Previously Pouton and his group have studied the influence of mixed glycerides (blends of mono-, di-, and tri-glycerides) on the phase behavior of ternary glyceride/surfactant/water systems, but only to a minor extent. As glycerides (alone or blended) are typically good solvents of drugs and they promote absorption of water and self-emulsification, it was of particular interest of the present study to find the appropriate glyceride/surfactant blends which are able to solubilize a large proportion of water [16]. These studies were carried out to obtain a comprehensive understanding of how phase behavior varies for different lipid-surfactant compositions as they are diluted with water. In addition to these investigations, the particle size distribution of the formulations after dispersion in water and the appearance of the emulsion droplets were analyzed as a function of different lipid composition. As lipid systems vary in the efficiency of self-emulsification, the particle size distribution was particularly necessary to evaluate the effect of composition on the emulsification properties and the influence of choice of oil and surfactant on drug incorporation. 531
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
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identified by making up 30-40 mixtures in the appropriate area of the phase diagram. Once an approximate map was available additional samples were prepared to determine boundary regions more precisely. Other phase boundaries of the phase diagram were also determined using the similar approach however, for practical reasons, given the number of phase diagrams investigated, in some cases the position of phase structures (lamellar/hexagonal etc.) was not determined. Phase behavior was examined using samples held at equilibrium for 48 h at 20 and 37 °C with the aid of a water bath (Ratek Instruments Pty. Ltd., Boronia, Victoria). Phase behavior was assessed initially by visual observation, classifying mixtures as one-phase or multiphasic (turbid) mixtures. LC phases were identified using an Allen viewer fitted with cross-polarizing filters, which were further identified as hexagonal or lamellar, whenever possible, based on the birefringence patterns observed under crossed polarized light of an optical microscope (Carl Zeiss, Axiolab, Jena, Germany) employing a magnification of 10-40× [18, 19]. To ensure uniformity, during the making each mixture was thoroughly blended with a Vm1 vortex mixture (Ratek Instruments Pty. Ltd., Victoria), and heated if necessary. Sample mixtures were weighed into and stored in glass tubes with water-tight closures (13×100 mm Pyrex, lined screw cap, Corning, United States).
I. Materials and Methods 1. Materials
Miglyol 812 (M812, medium chain triglyceride, C8-C10), Imwitor 988 (I988, medium chain mono- and diglycerides), Imwitor 308 (I308, 98 % medium chain monocaprylate) and propylene glycol (PG, 98 % pure) were supplied by Sasol Germany GmbH, Werk Witten, Germany. Capmul-PG8 (C-PG8, medium chain monocaprylate) was supplied by Abitec Corporation, Lloyd Hause, Janesville, WI, United States. Soybean oil (long chain triglycerides, 99 % pure) was obtained from Sigma-Aldrich Co, St. Louis, MO, United States. Maisine 35-1 (Maisine, long chain glyceryl monolinoleate) was provided by Gattefossé, Saint-Priest, France. The non-ionic surfactants used were polyoxyethelene-(20)-sorbitan monooleate (Tween 80, HLB-15) and polyoxyethelene-(20)-sorbitan trioleate (Tween 85, HLB-11), supplied by Sigma Aldrich Pty. Ltd., Castle Hill, New South Wales, Australia. 1 M HCl, which was diluted to obtain 0.1 M solution, was purchased from Merck (Darmstadt, Germany). All excipients were used as received without any further purification. Water used in this study was obtained from a Milli-Q water purification system (Sartorius, Göttingen, Germany).
2. Sample preparation and determination of ternary phase diagrams
3. Particle size determination
It was of particular interest to this study to determine the position of the phase boundary representing the maximum mass of water that can be solubilized by each glyceride/surfactant mixture. The phase boundaries of several phase diagrams were mapped out for the glyceride/surfactant and/or cosolvent with water, in some cases with phosphate buffer saline (PBS) and 0.1 M HCl (Table I) to find which combinations of mono-, di- and tri-glycerides, C-PG8 with Tween 80 (T80) or Tween 85 (T85) give rise to large regions of optically transparent mixtures (microemulsion regions) [16]. Samples for phase studies were prepared at three different stages such as primary mixtures, secondary mixtures and ternary mixtures at various weight ratios. Water was added progressively into each primary anhydrous glyceride-surfactant mixture. The transition from a clear to a cloudy solution as water was added indicated that the maximum solubilization had been exceeded [17]. For each phase study, a total of 80-100 mixtures were prepared to examine the whole phase diagram. Water uptake by glyceride-surfactant mixtures, to form water in oil (w/o) microemulsions produced transparent, apparently single phase regions, which were designated L2, using the usual terminology for the oil-rich phase. Initially the shape and size of the L2 region was
An understanding of the factors governing the particle size of resultant emulsions is important in designing any successful lipid formulations, particularly SEDDS/SMEDDS. In the current studies, emulsification performance of MCT and the blending of mono-, diand tri-glycerides were compared with long chain triglycerides (LCT) and similar product such as Maisine 35-1 (Maisine mono-glycerides: 32-52 %, di-glycerides: 40-55 %, and tri-glycerides: 5-20 %) [20] using surfactant T80. There are several techniques available at present to measure the particle size of dispersed systems. In this study, the Zetasizer 3000 (Malvern Instruments Limited, United Kingdom) was used by a combination of quasi-elastic laser scattering with photon correlation analysis to evaluate the particle size distribution (small droplets within 10-3000 nm) of the emulsions after dispersion in water The concentration used was selected to give a suitable obscuration value in the Zetasizer. A count rate of 50-500 Kcps was used to ensure accurate measurements (sample dilution was 1 in 1000, which did not alter the particle size). This analysis was performed on sample volumes of 1 mL using glass cuvettes. All measurements were performed at room temperature in triplicate, with 10 minute runs, using a 400 mm aperture. Prior to commencing the study, commercially available latex beads (Duke Scientific, Palo Alto, CA, United States) of known diameter (220 nm ± 6nm - NIST traceable, for reference standard) was subjected to size determination by the operating conditions of the instrument described above. Results were expressed as the Z-average mean which is the harmonic intensity-averaged particle diameter.
Table I - Components used to construct the equilibrium phase diagrams. Phase diagram
Figure
Oil
Surfactant
Aqueous phase
Medium chain triglycerides
1 2
M812 M812
T85 T80
Water Water
Medium chain glyceride blends
3 4 5 6 7
M812/I988 M812/I988 M812/I988 M812/I988 M812/I988
T85 T85 T80 T80 T80
Water Water Water Water Water
Medium chain mono- and diglycerides
8 9
I988 I988
T85 T80
Water Water
Medium chain mono-glycerides
10 11 12a 12b
C-PG8 I308 I308 I308
T80 T80 T80 T80
Water Water PBS 0.1M HCl
Cosolvent
II. Results 1. Correlation between self-emulsification and phase behavior
Using the equilibrium phase behavior studies, it is possible to obtain the mechanisms of self-emulsification process [21]. The phase regions in the current studies were named using conventional nomenclature system. In the ternary phase diagrams, represented in Figures 1-12, oil-continuous regions were denoted L2 (clear isotropic oily system), water continuous regions L1 (clear isotropic aqueous system) and the liquid crystalline phases were denoted LC. These LC structures were studied in detail (described further below) only for some of the ternary systems under optical microscope, which may contain either hexagonal or lamellar phases. For practical reasons no detailed attempt was made to quantify the volume fractions of the phases presented in the multiphasic mixtures.
PG
M812: Miglyol812, I988: Imwitor988, C-PG8: Capmul PG8, I308: Imwitor308, T85: Tween85, T80: Tween80, PG: propylene glycol and PBS: phosphate buffer saline. 532
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 1 - Triangular phase diagram of the M812/T85/water system (representing a typical LFCS Type II system) at (a) 20 and (b) 37 °C. L1 is water continuous phase, L2 is oil continuous phase, L1+L2 is two phase emulsion, L2+Lα is two phase oil and lamellar liquid crystals region, respectively. Identity and location of each phase is determined by polarized optical microscope. The dotted line denotes the area where lamellar phases were observed. Key: M812: Miglyol 812 and T85: Tween 85.
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Figure 3 - Triangular phase diagram of the M812:I988 (7:3)/T85/water system (Type II) at (a) 20 and (b) 37 °C. The broken line drawn near the water corner at 37 °C shows the three phase emulsion (L1+L2+Lα). When the liquid crystalline phases are denoted as Lα, these mixtures contained lamellar phases. Key: M812: Miglyol 812, I988: Imwitor 988, and T85: Tween 85.
Figure 4 - Triangular phase diagram of the M812:I988 (3:7)/T85/water system (Type II) at (a) 20 and (b) 37 °C. (L2+LC) represents a two phase system of liquid crystal dispersed in oil phase, and (L1+L2+LC) emulsion with liquid crystalline phase. When the liquid crystalline phases are denoted as LC these mixtures may contain either hexagonal or lamellar phases. The solid line denotes the area where a lamellar phase is formed. Key: M812: Miglyol 812, I988: Imwitor 988, and T85: Tween 85.
Figure 2 - Triangular phase diagram representing the M812/T80/water system (LFCS Type IIIA) at (a) 20 and (b) 37 °C. Water continuous phase is designated L1, oil continuous phase is L2 and liquid crystal phase “white birefringence” is LC. Key: M812: Miglyol 812 and T80: Tween 80.
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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 5 - Triangular phase diagram of M812:I988 (3:7)/T80/water system at (a) 20 and (b) 37 °C (Type IIIA systems). Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, two phase emulsion is (L1+L2) and liquid crystal phase “white birefringence” is LC, which contained lamellar structure. Key: M812: Miglyol 812, I988: Imwitor 988, and T80: Tween 80.
Figure 7 - Triangular phase diagram for the system M812:I988:PG (2:2:1)/T80/water system at (a) 20 and (b) 37 °C. Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, two phase emulsion is (L1+L2). Key: M812: Miglyol 812, I988: Imwitor 988, PG: Propylene glycol and T80: Tween 80.
Figure 6 - Triangular phase diagram of the M812:I988 (7:3)/T80/water system at (a) 20 and (b) 37 °C (Type IIIA systems). Oil continuous phase is designated L2, surfactant phase containing liquid crystal is (S+LC), and two phase emulsion is (L1+L2). Key: M812: Miglyol 812, I988: Imwitor 988, and T80: Tween 80.
Figure 8 - Triangular phase diagram of the I988/T85/water system at (a) 20 and (b) 37 °C. Different compositions of the formulation result in the formation of different phase structures. Oil continuous phase is designated L2, in presence of liquid crystal is L2+LC, two phase emulsion is (L1+L2). Key: I988: Imwitor 988, and T85: Tween 85.
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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
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Figure 9 - Triangular phase diagram of the I988/T80/water at (a) 20 and (b) 37 °C (Type IIIA). Oil continuous phase is designated L2, water continuous is L1, surfactant phase is S, and two phase emulsion is (L1+L2). Key: I988: Imwitor 988, and T80: Tween 80.
Figure 11 - Triangular phase diagram of the system I308/T80/water at (a) 20 and (b) 37 °C (Type IIIB), adapted from ref. 23. Oil continuous phase is designated L2, two phase emulsion is (L1+L2) and surfactant phase S. Both lamellar and hexagonal phases are present in the multiphase region of the phase diagram. Key: I308: Imwitor 308, and T80: Tween 80.
Figure 10 - Triangular phase diagram of the system C-PG8/T80/ water at (a) 20 and (b) 37 °C (Type IIIB). Oil continuous phase is designated L2, in presence of liquid crystal is L2+LC, two phase emulsion is (L1+L2) and the two phase emulsion in presence of liquid crystal is (L1+L2+LC). Key: C-PG8: Capmul PG8 and T80: Tween 80.
Figure 12 - Triangular phase diagram of the I308/T80/water system at 37 °C. (a) PBS (pH-7.5) and (b) 0.1 M HCl (pH-1.1) were used for the aqueous component. Oil continuous phase is designated L2, two phase emulsion is (L1+L2) and the two phase emulsion in presence of liquid crystal is (L1+L2+LC). Key: I308: Imwitor 308, and T80: Tween 80.
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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
For example (L1+L2) represents a turbid emulsion system in the phase diagram but the volumes and precise compositions of each phase were not investigated in detail. Typically (L1+L2) mixtures were formed close to the oil-water axis. Multiphasic dispersions of L1, L2 and LC phases also produced emulsions that had the same outward appearance as L1+L2 emulsion. It was possible to distinguish L1+L2+LC mixtures from L1+L2 when the Allen viewer revealed that LC was also present in the turbid mixtures. A clear strong birefringent LC observed adjacent to L2 regions was identified as L2+LC mixtures. L1+LC mixtures were mostly identified near the water-rich corner. In some of the phase diagrams an isotropic viscous gel-like phase was identified as Surfactant phase (S). This was considered to be a single phase rich in surfactant but with large amounts of water solubilized in it. These mixtures were difficult to distinguish from the L2 phase except looking at the viscosity.
Further dilution of this system with water produced liquid crystalline phases in combination with L2 or L1 and L2. The L2 region included a finger-like projection towards the water apex at 60-70 % surfactant (Figure 2b). This is a typical feature of SEDDS phase diagrams which is thought to correspond with rapid emulsification. 3.2. Medium chain glyceride blend Figure 3 depicts the phase diagram for M812:I988 (7:3)/T85/water system, where the oil component was a blend of M812 and I988 in the weight ratio 7:3. The anhydrous system solubilized a considerable mass of water, compared to the corresponding M812 system, as indicated by the extensive L2 region. Solubilization of water was pronounced at T85 concentrations between 40-50 %, up to 40 % water by weight at the maximum, and featuring the characteristic finger-like projection of the L2 region towards the water axis. The blending of M812/I988 at 7:3 ratios improved the performance of the LFCS Type II system compared to the previous system that contained M812 alone. This study suggests the substantial differences in the phase diagrams when the polarity of the oil phase is increased by the blends of mono-, di- and tri-glycerides with T85. It is important to note that M812 itself and the mixture of M812/I988 (7/3, w/w) are very poorly soluble in water. Figure 4 also shows the similar Type II systems with a different oil ratios of M812/I988 (3/7, w/w). The boundary of the L2 region of this phase diagram was slightly reduced in area in comparison with that shown in Figure 3. The large mass of water (approx. 35 %, Figure 4a) was taken up by the oil/surfactant (50/50, % w/w) anhydrous formulation. A slender (L1+L2+LC) phase at 20 °C and (L1+LC) phase at 37 °C (Figure 4b) was observed close to the water axis. Given the benefit gained by increasing the polarity of the oil phase with T85, it was appropriate to examine what effect the oil polarity had on LFCS Type III systems formed with hydrophilic surfactant T80. The replacement of T85 with T80 produced a significant change in the shape and extent of the L2 region, reflecting the affinity of T80 for water. These systems were Type IIIA systems, capable of producing very fine dispersions (< 100 nm, optically clear if higher concentration of T80 used). Mixtures of T80 with M812/I988 (3/7) were very good self-emulsifying systems which solubilized a large mass of water (Figure 5a-b). A transparent surfactant phase (S) was identified next to L2 region along the surfactant/water axis. This high viscous S phase contained hexagonal structure between 40 % & 50 % water. There was a bluish (transition between transparent and emulsion) phase observed at 37 °C towards the water apex within L2+Lα phase region (Figure 5b). The phase diagram for M812:I988 (7:3)/T80/water system also represents LFCS Type IIIA shown in Figure 6. This phase diagram can be compared with M812:I988 (7:3)/T85/water of Type II system (Figure 3) to see the effect of surfactant on the phase behavior. In fact, there was a significant difference to the water uptake towards the surfactant apex of the phase diagram (Figure 6a-b). At 37 °C, when the anhydrous mixture contained 70-80 % T80, the system was capable of absorbing an equal mass of water before a phase change occurred. With addition of more water, the two phase region S+LC (surfactant in equilibrium with liquid crystals) was formed in this system. Including PG in the oil phase (blended with M812 and I988) was also explored as a single glyceride component to increase the polarity of M812:I988:PG (2:2:1)/T80/water system (Figure 7). This system produced a large isotropic L2 phase region, which was reversed to L1 towards the water corner. Large (L1+L2) emulsion region was present along oil-water axis with addition of 20-30 % T80. However, above 30 % T80 there were large areas of mutual solubilization of the excipients and water. At extremely high surfactant concentration (9099 % T80) within L2 region, S phase was extended half-way towards the water axis. Another small (L1+L2+LC) region was present close to the water apex which was evident at 20 °C (Figure 7a) but disappeared to L1+L2 (emulsion) region at 37 °C (Figure 7b).
2. Characterization of maximum water uptake (L2/L1 phase)
The ternary phase diagrams constructed in the study indicate the phase changes which occurred when anhydrous oil-surfactant mixtures are mixed with water. Many of the systems were capable of absorbing considerable amounts of water, so that an extensive L2 region containing up to 55-60 % water. Figure 11 shows a typical example of the L2 phase that formed on dilution of a LFCS Type IIIB formulation. This shows the phase diagram of a simple but effective formulation comprising I308 and T80, which allowed the system to solubilize a large mass of water. However in this system, the L2 phase transformed into a transparent aqueous microemulsion or micellar system (L2 to L1, i.e., w/o to o/w microemulsions) [22]. These phases illustrate differences which are expected to influence the mechanism and efficiency of self-dispersion, which was only possible by determining the phase diagrams.
3. Introduction to the phase diagrams
The phase diagrams described in the studies depicted in Figures 1-12 were constructed at ambient (20 °C), and body (37 °C) temperature to investigate whether there were any substantial differences in phase behavior over the range ambient to body temperature. 3.1. Medium chain tri-glyceride The triangular equilibrium phase diagram for the M812/T85/water system at 20 °C and 37 °C is shown in Figure 1. Among the various phases formed by these three components, a clear and transparent oily region (dark grey L2 area) has been identified close along to the oilsurfactant side. This system represents an example of LFCS formulation Type II. Combinations of M812 and T85 containing between 30 % and 60 % produced SEDDS that have no water-soluble components. Figure 1b indicates that a maximum of 15 % water was solubilized by the anhydrous mixtures, i.e. at higher surfactant concentrations. On further dilution of the anhydrous systems with water, a phase change resulted in a distinct two phase region (L2+LC), which is typical for Type II systems (LC containing lamellar phase, Lα). The emulsification with non-ionic surfactant T85 in the system is thought to involve dynamic formation of lamellar liquid crystals at the oil-water interface. This would allow penetration of water into the bulk material by way of aqueous channels, causing an increase in interfacial instability, resulting in droplets being propelled from the inter phase. However the M812/T85 system is not as rapidly dispersing as related systems which include mono- and diglycerides. Figure 2 shows the ternary phase diagram for the M812/T80/ water system. The L2 region had a modest area, which indicated that relatively little water was solubilized by this system. In the L2 region, a maximum of 15 % water was solubilized at 20 °C when the weight proportions of oil and surfactant were equal. Solubilization of water was significantly increased to 28 % at 37 °C, which may indicate that emulsification is more efficient at 37 °C (Figure 2b). 536
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
3.3. Medium chain mono- and di-glyceride blend The phase diagram of the I988/T85/water system (Figure 8) provides information regarding the effect of mono- and di-glyceride on the phase behavior. Solubilization of water within the L2 region is moderate at most oil-surfactant anhydrous mixtures. Just the inclusion of MCT could have significant phase changes as evident by M812:I988/ T85 system (Figure 3). In the system with only I988 (Figure 8), the pointed finger-like region formed at approximately 50 % w/w glyceride/surfactant ratio. It is worth knowing that I988 is a polar lipid which was able to solubilize 15 % water itself in the absence of T85 (Figure 8a). Replacing T85 with T80 in the system resulted in more isotropic regions in the phase diagram shown in Figure 9. The L2 and S phases were clearly evident with reverse L1 phase. A finger-like projection in the L2 area was centered at the 50/50 ( %w/w) glyceride/surfactant ratio.
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on the phase behavior was determined by comparison of the relevant phase boundaries with those recorded on the corresponding diagrams constructed with water (Figure 11). There were some differences between the phase diagrams (Figure 11b versus Figure 12a-b) but in general the features recorded with water with preserved. In the alkaline buffer system, liquid crystalline (LC) phase was not existent with oil (L2) and water (L1) continuous phases at 37 °C (Figure 12a). Below the L2 region in the I308/T80/PBS system, the (L1+L2) emulsion was formed. In contrast LC was still present within the L1+L2 phase in I308/T80/0.1 M HCl system at 37 °C (Figure 11b). The location of the L2 phase regions in both systems were not significantly affected by the variation of pH. These systems will need to be investigated further to determine whether dilution in acid or buffered systems can have any effect on the solubilization and dispersion of the drug.
5. Effect of temperature on the phase diagrams
Ternary phase diagrams at two different temperatures (20 and 37 °C) were constructed only to determine the possible effects of temperature on the dispersion process. It was also of interest to compare the areas of various phase regions and the effects of temperature on liquid crystal formation. A size reduction of L2 phase with increase of the temperature was apparent in the phase diagram of M812:I988 (7:3)/T85/water system (Figure 3). Temperature increases also commonly decreased the area of regions which included liquid crystal. For example, the I988/T80/ water system (Figure 9b) had a reduced L2+LC phase region at 37 °C. The L2 phase region in the I308/T80/water system (Figure 11) was affected significantly by temperature variation. A temperature increase to 37 °C eradicated the wax formation in the oil (I308) corner due to melting of the mono-glyceride content (Figure 11b). On the other hand, the area of L1 phase region was increased at 37 °C. This appeared to be due to a reduction in the viscosity of the S phase which resulted in the assignment of a larger L1 phase region. In common with other systems, the LC phases melted as temperature was raised so that the three phase region (L1+L2+LC) disappeared at 37 °C and became L1+L2. I308 (waxy mono-glycerides) was able to solubilize almost 10 % more water at 37 °C than at ambient temperature which indicates that self-emulsification is likely to be more efficient at body temperature.
3.4. Medium chain mono-glyceride C-PG 8/T80/water represents the LFCS Type IIIB system, which shows the effect of using only mono-glyceride with hydrophilic surfactant (Figure 10). This composition, which is highly water-soluble in nature, produced a different shape of L2 phase, presented as hooklike dark grey area. With C-PG8, at high concentrations of surfactant (above 70 %), there was substantial solubilization of water, particularly at 37 °C (Figure 10b), although the uptake of water at lower surfactant concentrations was not as significant as for other Type III formulation systems (Figure 5b). Replacement of the C-PG8 with I308 in the glyceride (oil) corner created a remarkable change in the extent of water absorption by the L2 phase (Figure 11). The essential difference between C-PG8 and I308 is one extra hydroxyl group per molecule in I308. A wide range of clear transparent mixtures covering a surprising variety of mixture compositions was evident in Figure 11b. In this regard, I308 is a rather unique excipient which may be very useful for lipid formulations. In addition, the isotropic L2 region was extended up to S and L1 phases. A bluish transparent phase was observed within L2 phase at very high water contents in regions that were thought to be colloidal in nature (Figure 11b) [23]. At concentrations of T80 below 50 %, a phase change occurred when excess water was added, producing L2+LC systems (Figure 11a). The 50 % T80 system dispersed to produce a fine emulsion characteristic of SMEDDS, though it should be noted that this precise formulation has not been investigated by other authors, and it may have unique properties being so rich in mono-glycerides. Although I308 was a useful excipient which promoted absorption of water and efficient emulsification, it had the disadvantage that the pure mono-glyceride was semi solid waxy at ambient temperatures. For this reason, the phase diagram of the I308/T80/water system was determined in detail at 20 °C in the vicinity of the I308 apex to identify practical problems that might occur due to crystallization of waxy mono-glycerides. The region in which crystals were detected is displayed as the dark gray area in Figure 11a. This data was obtained by keeping the samples at 20 °C for 15 days before making an assessment by visual inspection. The waxy phase melted between 20 and 37 °C. This data indicated that the use of I308 as the single oily component may not be practical at I308 concentrations above 40 % because the anhydrous materials may become segregated if crystallization of mono-glyceride occurs on storage.
6. Investigation of liquid crystalline structures using optical polarizing microscopy
The presence of liquid crystalline material in some of the formulations was initially identified in the vials with the samples viewed between crossed polarizers (Allen viewer) to probe the structure of the LC phases. The hydrated samples with different water content were then examined under a polarizing light microscope (samples placed between micro slides and cover slips) to detect the presence of optically anisotropic phases (lamellar and hexagonal phases). The textures of the hexagonal and lamellar liquid crystalline materials when viewed by polarizing microscopy are distinctly different. In the phase studies, a large anisotropic area containing lamellar (Lα) phase (Figure 13a-b) exists in many of the ternary glyceride/ surfactant/water systems (Figures 1, 3 and 11) at water contents ranging from 15 % to as much as 90 %, and these phases are perhaps more prevalent at 20 °C. The Lα phase consists of bilayers separated by water, forming the comparatively low viscosity Lα liquid crystalline phase, the viscosity being another indicator of structure. In M812:I988 (3:7)/T80/water system (Figure 5), hexagonal liquid crystal (H1) and Lα liquid crystal phases were successively formed within Surfactant (S) region. It was thought that cylindrical micelle structures were closely packed in a hexagonal array in the H1 phase which could be identified by its optical microscopy pattern (Figure 13c-d). The hair-like texture of hexagonal phase (H1) was seen at 40 % to
4. Influence of pH on the phase behavior
Dispersion of lipid-based formulations in vivo takes place either in the gastric or intestinal fluids, rather than in pure water. In order to investigate whether the pH of the aqueous medium influences the phase behavior of the I308/T80 formulation, phase diagrams were investigated using phosphate-buffered saline (PBS, pH-7.5, Figure 12a) and 0.1 M hydrochloric acid (pH-1.1, Figure 12b) at 37 °C. The effect of pH 537
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The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
Figure 14 - Emulsion droplet diameter of dispersions formed by selfemulsification systems with M812/I988 (7/3, % w/w), SOB/I988 (7/3, % w/w), M812/Maisine (7/3, % w/w) and SOB/Maisine (7/3, % w/w) each in combination with Tween 80, as a function of surfactant concentration. Formulations were emulsified with water at room temperature for 10 min and then analyzed by photon correlation spectrometer (PCS). Data are mean ± SD (n = 3). Key: M812: Miglyol 812, I988: Imwitor 988, and SOB: soybean oil.
Figure 13 - Representative photomicrographs of (a) & (b) lamellar liquid crystalline phase structures, and (c) & (d) hexagonal liquid crystalline structures. These images were taken using samples of I308/Water (60/40, % w/w) and T80/Water (50/50, % w/w) between cross polarizers under light optical microscope. Key: I308: Imwitor 308, and T80: Tween 80.
low concentrations the emulsions were coarse at surfactant concentrations lower than 40 % w/w. A few studies have reported similar trends in droplet size with increase in surfactant concentration for various self-emulsifying systems [25]. Small particle size did not necessarily correlate with rapid emulsification. At high surfactant contents > 7080 %, the dispersions formed were essentially transparent but these dispersions often took much longer to reach completion. Various compositions of M812:I988 (7:3) and T80 appeared to emulsify quite rapidly and produced fine dispersions at room temperature. The most efficiently dispersing systems (dispersed ≤ 1 min in water) were formed at 30 to 50 % surfactant concentrations, producing dispersions with particle sizes less than 300 nm. M812:I988 (7:3)/T80 systems containing above 90 % T80 took > 5 min to disperse. The replacement of MCT with LCT (SBO) to produce the systems affected considerably the efficiency of emulsification. Generally mixtures containing SBO produced more turbid, coarser dispersions. When these systems were analyzed using the Malvern Zetasizer the apparent mean particle size was relatively high, close to or in excess of 1 μm. The apparent particle size distributions and dispersion times of M812:Maisine (7:3)/T80 and SBO:Maisine (7:3)/T80 formulations were found to be similar, producing sub-micron dispersions at surfactant concentrations above 30 %. The M812:Maisine system produced significantly finer emulsions but a limited particle size differences between the two systems were not likely to determine the differences between their performances. Thus in the long chain mixed glycerides there appeared to be little advantage gained by including MCTs.
50 % water in surfactant-water binary mixtures (T80/water composition in Figure 11). When 5 % I308 was added to the binary surfactant-water system, the H1 phase reverted to a multiphasic region (containing Lα) as shown in Figure 11a. An optically anisotropic liquid crystalline region in binary mixtures of I308 with 30-60 % (w/w) water formed lamellar structures (Figures 11a and 13a). An increase in temperature (to 37 °C) strongly reduced the presence of the Lα phase and transforms it into a fluid isotropic L2 phase. However, I308 did not appear to exhibit the classical phase sequence: L2 (reversed micellar), HII (reversed hexagonal), QII (reversed cubic), and Lα (lamellar). This sequence is often observed upon hydration of poorly soluble amphiphilic molecules [24].
7. Effect of formulation on the efficiency of self-emulsification
A series of mixtures comprising medium chain glycerides and long chain glycerides with hydrophilic surfactant T80 were prepared and their self-emulsifying properties were studied using visual examination and particle size analysis. T80 is a popular choice of surfactant for formulation of SEDDS/SMEDDS, so it was of interest to compare the efficiency of the systems formulated with T80. Initially, anhydrous glycerides and surfactants were mixed in different proportions (10-100 % w/w) at room temperature so that a range of surfactant concentration could be investigated. One hundred milligrams formulation was introduced into 10 mL (1 in 100 dilution factor) of water in a glass beaker at room temperature, and the contents were agitated gently with a glass rod. The tendency to emulsify spontaneously and also the progress of emulsion droplets were observed against time. In this way, it was easy to distinguish between formulations that formed microemulsions, which were clear or slightly blue translucent dispersions, and formulations that formed crude emulsions on dispersion. An estimation of the time required for complete dispersion in water at ambient temperature was noted in triplicate. According to the visual inspection, < 5 s represented spontaneous dispersion whereas > 1 and > 5 min indicated the slow and longer dispersion times for the samples, respectively. Figure 14 illustrates the influence of the chain length (MCT vs LCT) over a wide range of surfactant concentration. For M812/I988 (7/3) and SBO (Soybean oil)/I988 (7/3) w/w, it is evident that the size of the emulsion droplets decreased with increase in the concentration of T80, but in a more marked manner for the long chain glycerides. At
III. Discussion
A critical characteristic of a successful lipid formulation is its ability to emulsify rapidly and disperse well in the aqueous media. Currently, many pharmaceutical excipients are not miscible themselves and poorly dispersible as they are diluted in aqueous media, which is creating a real challenge in terms of developing formulation. The ternary phase diagrams constructed using three component systems in the current research were adequate in determining phase transitions and developing optimal lipid formulations. However, in some cases, such as the identification of the phase structures of lamellar and hexagonal phases, a great deal more work would be required to pin-point the exact phase boundaries. These phase changes between different liquid crystalline phases could be identified by optical microscope, but were squeezed into a relatively small area (Figures 1-12). It is well known that self-emulsification is specific to the characteristics of the oil/surfactant affinity, surfactant concentration and the 538
The influence of the ratio of lipid to surfactant and the presence of cosolvent on phase behaviour during aqueous dilution of lipid-based drug delivery system K. Mohsin, C.W. Pouton
temperature at which self-emulsification occurs [7]. From the results of the current studies it was satisfying to observe that the inclusion of mono- and di-glycerides in the formulation systems led to increased efficiency of emulsification. However, to select any successful formulation, understanding of the behavior of mono-, di- and tri-glycerides and either hydrophilic or lipophilic surfactant when blended together is considered to be an initial step. Equilibrium phase studies can give a clear picture of how the excipients mix remains miscible, by identifying mixtures which can tolerate small changes in water content, such as might arise during encapsulation or drying of the manufactured product, as the capsule reaches its final hydration state. In the current studies, MCT oil with mixed mono- and di-glycerides were predominantly used for the design and the assessment of selfemulsifying formulations. T80 (HLB=15) and T85 (HLB=11) were chosen deliberately as a pair of chemically related materials, which represent a water-soluble and water-insoluble surfactant, respectively. They are known to have low toxicity, at least in the case of T80, and have been used popularly in many pharmaceutical products. The inclusion of mixed glycerides improved the quality of the emulsions reported in previous studies. It was reported that a selfemulsifying formulation of a lipophilic drug WIN 54954 with a combination of MCT (Neobee M5)/ non-ionic surfactant (Tagat TO)/ drug (40/25/35, % w/w), emulsified rapidly upon gentle agitation at 37 °C producing emulsions with mean droplet diameter of less than 3 µm [21]. In the current studies, it is also observed that mixed mono-, di- and tri-glycerides with surfactants have a higher tendency to uptake large amount of water and form emulsions of small particle size. The blending of tri-glycerides with mixed mono-and di-glycerides had a beneficial effect of improving water solubilization. In addition, the inclusion of PG in mixed glycerides produced systems with extensive amounts of water solubilization (Figure 7). Extensive water absorption is generally associated with rapid emulsification and fine particle size [2]. Phase diagrams of T80 with I308 in particular were found to have very large regions which are optically transparent (Figure 11). This transparent region could include L2 (oil-continuous) and L1 (water-continuous) phases which are microemulsions containing a considerable variety of microstructures [22, 26]. Many of these systems were also rich in surfactant, and could be referred to as “surfactant phase-S” with large masses of co-solubilized water and oil. The I308/ T80 system is a highly promising formulation since a large area of the phase diagram is covered by well emulsified systems. The work in this research has also shown that the emulsification properties of mono-glyceride/di-glyceride/tri-glyceride/Tween-based self-emulsifying systems are greatly dependent on the composition, as more hydrophilic mixtures showed a higher tendency to solubilize water and form emulsions of small particle size. It was evident that at very high concentration of surfactant (70-90 % w/w), microemulsions were formed with most of the glycerides (oils). These systems tended to be denser than water and dispersed slowly after the bulk material had sedimented. At lower concentrations of surfactant (20-60 % w/w) the dispersions were turbid, although they often self-emulsified in an efficient manner. In addition, these systems were found to be temperature sensitive in the temperature range 20~37 °C. Like most changes observed with temperature in the phase diagrams, this was likely to be due to the progressive reduction in solvation (i.e. hydration) of the surfactant ethoxylate chains which occurred as the temperature was elevated. Within the scope of the present study, LFCS Type II formulation systems were modeled by blends of glycerides and T85. Blends of M812 and T85 self-emulsified within a minute but were not a good system to solubilize much water (L2 phase region). However, the solubilization was improved significantly by blending of tri-glycerides with mixed mono- and di-glycerides. This is in agreement with similar unpublished work by Pouton and colleagues using the surfactant Tagat TO, which is also an ethoxylated trioleate ester with an HLB
J. DRUG DEL. SCI. TECH., 22 (6) 531-540 2012
close to that of T85. Type IIIA formulations were formed by replacing T85 with T80. They had improved emulsification properties and comparably reduced particle size. In these systems the medium chain mono-, di- and tri-glycerides could be replaced by long chain mono-, di- and tri-glycerides but with increase in particle size. The rate of emulsification and the particle size of the dispersed formulation are the key descriptors of the efficiency of self-emulsification process, though according to the most studies it was considered that a visual inspection of rate of emulsification was sufficient to compare formulations. I308/ T80 system could be considered to be a Type IIIB SMEDDS, but it may represent a new class of Type IIIB formulation which can have a high content of medium chain mono-glyceride, for example the system 50 % I308/50 % T80. Generally, rapidly emulsifying Type IIIA & IIIB formulations were efficient self-emulsifying systems with regard to particle size, and resulted in emulsions in the submicron range on aqueous dispersion. Some classes of lipids were able to form viscous liquid crystalline structures (lamellar/hexagonal) in excess water (Figure 13). The ability of alkyl glycerates to form reverse hexagonal phases was recently reported by Boyd et al. [27] There has been increasing interest in the potential pharmaceutical utility of liquid crystalline structures formed by polar lipids and or surfactants in excess water [28, 29]. The viscous hexagonal liquid crystalline phases offer considerable scope for drug delivery applications. They are of particular interest, as their structures provide a diffusion barrier to drug release when the drug is incorporated into the matrix on formation; thus they have potential as sustained-release matrices [30]. The effect of liquid crystalline structure on drug absorption after oral administration of liquid crystal-forming lipids is not yet clear. * Overall the current studies confirmed that mixed glycerides are good self-emulsifying systems, which can promote absorption of water and rapid dispersion. As they are good solvents for drugs, their attributes can be utilized advantageously to fit drug delivery challenges (particularly water insoluble drug), making lipid-based drug delivery systems a successful approach. Design rules for the choice of oil, surfactant, as well as the surfactant ratio for maximum solubilization may be established by the construction of phase diagrams. Amongst the LFCS systems the most efficient formulations were produced by ethoxylated sorbitan ester in combination with I308, which were categorized as Type IIIB. It was also possible to formulate good SEDDS with T80 as long as I988 or Maisine 35-1 were included.
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Acknowledgements The author is thankful to Dr. Michelle Long for her assistance during this study.
Manuscript Received 1 February 2012, accepted for publication 3 April 2012.
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