Formation And Characterisation Of Novel Microemulsions Biology
Micelles and microemulsions of dimethyledodecyleaminopropanesulfonate Dodecyletrimetyleamoniumbromide and Sodiumdodecylesulfate formed in water /menthol or in eutectic system were investigated using laser light and small angle neutron scattering. The size of the (micelles/microemulsion) system was concentration of surfactant and / or amount of oil dependent. Noticeable increase in size of system was observed in case SDS/menthol (4:10) and SDS/Eutectic (7:10). The results obtained by SANS showed similar trend in size however, the values were bit less than the one obtained by light scattering and the non-spherical characteristic was increased in surfactant/ Eutectic 2%, surfactant/ Eutectic 4%, surfactant/ menthol, irrespective of surfactant used.
The term microemulsion was used for the dispersion of two immiscible liquids such as oil and water which was stabilized by an appropriate amount of surfactant by Hoar and Schulman in 1943. The dispersion is isotropically clear, thermodynamically stable, low viscous and normally transparent. It is now-a-days widely used in pharmaceutics as a drug carrier.  In addition to surfactant, co-surfactant (in some cases) is also used for the stabilisation of the emulsion, which is short to long chain alcohol, depending upon the conditions. There can be three types of microemulsions, oil/water, water/oil and bi-continuous. The composition of microemulsions depends upon the properties of surfactants and oil used. The interfacial tension of microemulsion is very low and their size ranges from 10 -200 nm. 
Microemulsions have attracted much attention in the last few years in term of their drug delivery as a potential candidates. [6-13] It is due to their transparency, high solubilising capacity of drug, ease of preparation, long-term stability, and the ease in sterilization by filtration.  Further to it, the thermodynamic stability of microemulsions offers advantages over unstable dispersion, such as traditional emulsions and suspensions, because they can be prepared by little energy input (heat or mixing) and has longer shelf life. [15-17]
Water in oil and oil in water has shown to enhance the oral bioavailability of drugs although the mechanism of absorption enhancement is not yet established.  The drug delivered through such fluids is protected against enzymatic hydrolysis, its absorption is enhanced due to surfactants induced membrane fluidity and thus permeability changes.  Formulation with suitable recipients/ vehicles may also improve delivery of labile (peptides or proteins) and poorly soluble drugs.  We can say that such systems are capable of carrying and delivering almost every type of drug, safely to the target. [21-26] However, such useful properties of the microemulsion are linked to the intermolecular interactions which depend upon, in addition to other parameters, over their composition, size, oil used etc. Therefore, a number of techniques have been developed and used to characterize microemulsion. These can be macroscopic measurement, viscosity, conductivity measurement and electrical methods, dynamic and static light scattering and small angle neutron scattering. [27-34] Up to now both the structure and the mechanism of formation is not well defined nor are the forces involved in the process of formation / drug solubilization recognized. Therefore, our objective is to explore such a useful system in detail by estimating the phase area of these novel microemulsions and determine their size by light and neutron scattering techniques. It is hoped that in this way we will not only be able to measure the size and shape of microemulsions but can also establish their structure.MATERIALS AND METHODSMaterials
Dimethyledodecyleaminopropanesulfonate (DDAPS) (97%) was purchased from Fluka Biochemica, Dorset, UK. Dodecyletrimetyleamoniumbromide (DTAB) and Lidocaine were obtained from Sigma, Dorset, UK. Sodiumdodecylesulfate (SDS) a biochemical grade was purchased from BDH, UK. L-menthol was purchased from Alfa Aesar, UK. All the chemicals were used as obtained from the supplier/ manufacturers.Estimation of Aqueous solubility of menthol and eutectic mixture of lidocaine/menthol
The aqueous solubility of both menthol and eutectic mixture of lidocaine/menthol was determined through UV measurements and samples were prepared by saturating the water with menthol and eutectic mixture and then making the dilutions till the solution became unsaturated.Samples Preparation
The micelles samples were prepared by dissolving DDAPS, DTAB and SDS surfactants in deionized water while keeping the concentration of surfactants well above (0.2-1.0% w/w) CMC. For concentrated microemulsions formation, a mixture of surfactant (40% DDAPS, 40% DTAB or 20% SDS), menthol / eutectic mixture of menthol and lidocaine (70/30) in water was heated at 343K in closed container and then allowed to cool up to 298K. The amount of the surfactant used was fixed in such a way that the mixture was able to solubilise required amount of eutectic mixture and menthol. The dilute samples of micelles and microemulsions were prepared by sequentially diluting the freshly prepared concentrated samples of both microemulsions and micelles to required surfactant (0.2-1.0 % w/w) concentrations.Refractive Index Measurement
The refractive index increment of the material, having solubility around 0.03%, was measured using laser light source having wave length of 632 nm, over the high precision Abbes (60/ED) refractometer supplied by Bellingham and Stanely, Ltd, Sevenoaks, UK. The specific refractive index increment (dn/dc), required for Total Intensity Light Scattering analysis was then calculated from plot of R.I vs concentration of microemulsions.
Photon Correlation Spectroscopy and Total Intensity Light Scattering Measurements (TILS)
The photon correlation spectroscopy and total intensity light scattering was performed on two different instruments, Malvern 4700c light scattering instrument, supplied by Malvern instruments Ltd, Worcester UK, which can perform both photon correlation spectroscopy and total intensity light scattering measurements simultaneously, while the second one was DAWN EOS/QELS, supplied by Wyatt Technologies, USA, installed with a software, Astra for windows to do particle size analysis. At this instrument, photon correlation spectroscopy and total intensity light scattering measurements can be carried out separately. In all the surfactants samples the ratio of scattering intensities for TILS and diffusion co-efficient for photon correlation spectroscopy at an angle of 450 and 1350 were always in the range of 1.00 ±0.05 and hence rest of the measurements were made at 900.Small Angle Neutron Scattering
Small Angle Neutron Scattering, SANS experiments were performed using LOQ diffractometer at pulsed neutron source ISIS. LOQ diffractometer uses neutrons of wavelength 2.2-10 A, simultaneously by time of flight, with 64364 cm2 detector at a distance of 4.1 m from the sample. The measurements were made at surfactant concentration of 5% of the samples which were held in quartz sample holder of thickness 2mm. The temperature for all the samples was kept at 25.0 ± 0.1 0C.The data were recorded in the range of 0.009-0.2A-1 Q.RESULTS AND DISCUSSIONRefractive index increment
The results obtained for refractive index of the DDAPS and its microemulsions with menthol and eutectic mixture in water is plotted in Figure 1 as a function of concentration. The figure shows that the refractive index increases linearly with the concentration of surfactant / microemulsions of menthol and eutectic mixture, showing that the system is quite homogeneous in the investigated range. The slope of the line (dn/dc) for the system is listed in Table 1.The same procedure was adopted for other systems and they also showed a linear dependence of refractive index over concentration. The dn/dc obtained from the slope of such plots is reported in Table 1. The table indicates that the refractive index increment is high for DTAB system as compare to others, irrespective of micelles or microemulsions in menthol / eutectic mixture.Total Intensity Light Scattering
The mean intensity of scattered light was obtained at 90o (S90) by employing Equation (1), after making the correction due to scattering / reflection of light by thermostatic bath and sample cell etc.
Here Ssample, Swater and Stoluene stand for Iscattered/ Io for microemulsion, water/ solvent and standard, respectively and Io stands for intensity of incident light.
The results obtained in this way are plotted as a function of concentration of surfactants in Figure 2. The linear trend of scattering intensity with the concentration of surfactants suggests that the size does not depend upon the investigated range of concentration and hence allows the use of Debye equation to determine the micelles` size. The results so obtained are listed in Table 2. The table indicates that micelles` size varies in this order DDAPS> SDS> DTAB which may be due to difference in molecular mass of the surfactant used. This means longer the chain length bigger the size of micelles is; which is according to expectations.
The Rayleigh scattered ratio, R90 of each microemulsion sample with respect to toluene was calculated according to Equation (2)
Here the R90 of toluene was taken equal to 1.35-10-5 cm-1 for λ=632.8 nm at 25 0C.  Results obtained in this way are displayed in Figure 3 as a function of concentration. The figure shows a linear dependence of R90 over the concentration of surfactants.
According to Debye equation, light scattering from a dilute micellar solution, at an angle θ, is described by the relation. 
Here, C and Ccmc is the total concentration (in g cm-3) of surfactant and microemulsion, respectively. -Rθ is excess Rayleigh ratio, B is second varial coefficient. K is an optical constant calculated as
Where n is the refractive index of solvent; dn/dc is the refractive index increment of the sample; NA is the Avogadro's number and λ is the wave length of incident light.
The mass of aggregates and aggregation number can be calculated as
The linear dependence of K(C-Ccmc)/-Rθ over the concentration of surfactants concludes that it obeys Equation (2) (Figure 4). The molecular mass of the micelles obtained from the intercept of these plots is listed in Table 2. The aggregation numbers of surfactants obtained from these values and employing Equation (6) are listed in Table 2. The table indicates that micelles size and aggregation number vary in the following way DDAPS> SDS>≈ DTAB. The reason for such trend can be the same as in case of micelles size.Dynamic Light Scattering
The dynamic light scattering also known as photon correlation spectroscopy has been employed to measure the size of the droplets. For the purpose, Stokes-Einstein equation (Eq (7)) has been used and the hydrodynamic radius (hR) of the droplet has been determined
Here k is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the continuous phase and D is the diffusion coefficient.
At infinite dilutions, the hydrodynamic radius of micelles (assuming spherical aggregates) was found to be 2.84 nm, 2.49 nm, and 2.16 nm for DDAPS, SDS and DTAB, respectively (Table 3). The results obtained are also plotted in Figure 5 and conclude that the hydrodynamic radius of DDAPS remains almost constant over the investigated concentration (0.2% to 1%) range. On the other hand the micelles size of DTAB and SDS was decreased and the diffusion coefficient was increased with the increase in concentration, which might be due to the strong repulsive forces among the micelles. Microemulsion Formulated using DDAPS.
The relative light scattering intensity measured at an angle of 900 was plotted over the range of surfactants concentration which showed a linear dependence over the concentration as in case of its pure micelles. The results indicated that though there was an increase in intensity in both cases but much lower than expected for microemulsions. Such behaviour is attributed to greater dilution of DDAPS/Eutectic microemulsion and little amount of oil left in micelles to show any swelling and partially due to small increase in aggregation number of DDAPS in the microemulsion systems.  The molecular mass of aggregates and their aggregation number obtained from the plots of R90 vs concentration are listed in Table 3. It was noticed that the aggregation number of micelles increases up to small extent with the concentration due to the fact that small molecular volume of oil cannot bring any significant change in micellar size. 
Figure 6 represents the variation in hydrodynamic size of microemulsion prepared in menthol / eutectic mixture, keeping the DDAPS to oil ratio as 10:1 and measured through dynamic light scattering. The results indicated that the size in both (menthol (2.87 nm) /eutectic (2.89 nm) cases is comparable to the pure micelles but increase regularly and almost equally with the amount of different dispersed system.Microemulsions formulated with DTAB
The variation in scattering intensity and Rayleigh ratio of DTAB of microemulsions droplets containing menthol (2:10) and eutectic mixture of menthol and lidocaine as an oil (2.5:10) were calculated and plotted versus concentration. It was noticed that the slope of scattered intensity and Rayleigh ratio was different for both the systems, indicating that the variation in degree of interactions and size with concentration was different for both the systems. Further to it, these parameters are also noticed to be affected by shape of droplets or micelles which are spherical in this case. Molecular mass of aggregates obtained through Debye equation and aggregation number obtained from the data are listed in Table 3. The table indicates that the size is greater in this case as compared to earlier system due to incorporation of greater amount of oil.
The hydrodynamic radius obtained through dynamic light scattering measurement is plotted in Figure 7 as a function of surfactant concentration. The figure shows that the size of droplets of (DTAB/Menthol) microemulsion is 3.02 nm and that of DTAB/eutectic microemulsion is 3.07 nm for dilute system and increases with the increase in concentration of surfactant. It is due to the fact that by increasing the concentration the growth of micelles takes place and the aggregation number increases as observed by others for different systems.Microemulsions formulated with SDS
The scattering intensity of SDS/Menthol (10:4) and SDS/Eutectic (10:7) mixture was plotted as a function of concentration. The scattered light intensity was observed to increases with the concentration indicating the variation in size and interactions in the micelles/ microemulsion. Further to it, the intercept of both the curves was different, indicating different size/ aggregation number for the systems. It was noted that the scattered intensity (Figure 8) increases smoothly with concentration which is the property of microemulsion and contrary to micelles. 
The molecular mass of microemulsion obtained through light scattering and aggregation number calculated from the curves R90 are recorded in Table 3. Since more oil has been incorporated in the micelles for both cases, the size has been increased for SDS/menthol microemulsions (3.14 nm) and SDS/Eutectic (3.21nm). Further the size increases with the concentration of the surfactant added which is due to change in aggregation number (Table 3).Analysis of SANS data
When a beam of neutrons is targeted over a particle/ droplet it is scattered and the incoherent differential scattering cross section (d∑/dð›º) for a system of mono-dispersed interacting particles can be expressed as (40-41]
) 2 V2 [F2 (Q)] + [F (Q) 2 [S (Q) - 1] + B
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