Effect of drug aceclofenac on physicochemical properties of mixed micellar systems

In this article, the effect of the drug aceclofenac (ACF) on the properties of three mixed micellar systems are studied. The three systems were pluronic L64 + F127 (nonionic-nonionic), pluronic L64 + CTAB (cetyltrimethyl ammonium bromide), (nonionic-cationic) and L64 + SDS (Sodium dodecyl sulphate), (nonionic-anionic) combinations. The physicochemical parameters were characterized by different techniques such as UV visible spectroscopy, FTIR, conductance, DLS, and SEM. The presence of ACF affected the nonionic-ionic mixed micelles more than the nonionic-nonionic group as evidenced by UV spectroscopy. From the DLS measurement, it was observed that ACF enhanced the size of the single micelle of pluronic L64 from 98 to 168 nm. The size of the cationic mixed micelle with ACF displayed 329 nm and the anionic mixed one showed 291 nm suggesting enhanced entrapment efficiency of their mixed micelle compared to the single micelles. The size was also reconfirmed by SEM analysis. From the conductivity measurements of the two nonionic-ionic micellar systems, the counter ion binding constant β, and the thermodynamic parameters ΔG, ΔH, and ΔS were determined. The negative value of ΔG infers spontaneous binding between ACF and ionic mixed micelles. Ionic mixed micelles are more effective than nonionic pair. ACF has more spontaneous binding with anionic mixed micelle compared to cationic. The drug entrapment efficiency is better in mixed micelles than in single micelles. Ionic mixed micelles are more effective than nonionic pair. ACF has more spontaneous binding with anionic mixed micelle compared to cationic. The drug entrapment efficiency is better in mixed micelles than in single micelles.


Introduction
Pluronic block copolymers are known to offer phase behavioural properties. Hence, they are used extensively in applications of drug delivery. They encompass hydrophilic polyethylene oxide (PEO) and hydrophobic polypropylene oxide (PPO) blocks in a single moiety. These groups are arranged in a structure such as (PEO) n (PPO) m (PEO) n . They are available in wide ranges depending on various PO/ EO ratios as well as molecular weights. Because of their amphiphilic behaviour, they have attracted researchers from various fields, especially for the application of solubilization and delivery of hydrophobic drugs [1,2].
The single micellar pluronic copolymer was known to dominate the drug delivery field until recent years. But currently, binary systems have started gaining importance. The deficiencies of a single micellar system, such as larger particle size, low drug loading capacity, and low stability are overcome through the use of mixed micelles of different polymers [3]. For example, Doxorubicin loaded in pluronic F127 and L64 micelles were the first of their kind to get approval for clinical tests used in cancer chemotherapy [4]. Vakil and Kwon prepared mixed micelle using 1,2-distearoyl-S n -glycero-3-phosphoethanolamine-N-methoxy poly(ethylene glycol) for drug amphotericin B which produced smaller sized thermodynamically stable and better-incorporated micelles than PEG 5000 -b-PCl x [5]. Mixed micelle structured by Gao et al. constituted from pluronic P105 and d-α-Tocophenyl polyethylene glycol 1000 succinate provided better solubilization efficiency and a stable system for drug camptothecin [6].
Pluronic P105 and L101 were used for mixed micelles to incorporate Paclitaxel (PTX) which was used for multidrugresistant tumours [7]. The pluronic binding micelles have been used to enhance the in vitro cytotoxicity and blood circulation time of PTX compared with Taxol ® [8]. A successful combination of pluronic F127 and pluronic L121 was prepared for Sudan (3) dye displayed 10 times more solubilization capacity, stable, and smaller sized micelles compared to that prepared from a single micellar system of F127. In another study, pluronic L81 was mixed with two pluronic surfactants viz. hydrophilic F127 and pluronic F87 to get highly unstable systems with turbidity throughout the experimental procedure. Hence, this work was undertaken to prepare three different combinations of mixed micellar systems, pluronic L64-pluronic F127 (nonionicnonionic), pluronic L64-CTAB (nonionic-cationic), and pluronic L64-SDS (nonionic-anionic), and check their various physicochemical properties in presence of the drug aceclofenac.
The mixed micellar systems generally display superior physicochemical properties compared to single micellar systems. Also, the properties can change due to the presence of certain additives. Hence, the change of certain properties of three mixed micellar systems due to the addition of ACF is investigated in this work.

Materials
All the chemicals used were of analytical grade and were used as received. Surfactants pluronic L64, Pluronic F127, CTAB, and SDS were procured from Sigma Aldrich supplied by Subra scientific company, Pondicherry. Drug Aceclofenac was obtained as a gift sample from M.M.C. Healthcare, Chennai. All aqueous solutions were prepared using double distilled water.

Preparation of mixed micellar systems
Stock solutions of pluronic L64 (17 mM) and F127 (0.397 mM) were prepared using double distilled water under cold conditions (4 °C). They were left at the temperature for 24 h. The stock solution of 50 mM CTAB and 80 mM SDS was prepared using distilled water. The stock solutions of pluronic L64, pluronic F127, CTAB, and SDS were taken in a 1:1 ratio for three mixed micellar solutions. Pluronic L64-pluronic F127, pluronic L64-CTAB, and pluronic L64-SDS were mixed. Different concentrations were used for different experiments. The mixed micellar solutions were allowed to equilibrate for at least 12 h before the characterization to allow the complete formation of aggregated structures.

Preparation of drug sample
About 0.05 g of aceclofenac was solubilized in 15 ml methanol. Out of which, 5 ml was evaporated to dryness. To the evaporated mass, 10 ml phosphate buffer pH 7 was added. It was then made up to 100 ml in a standard measuring flask. The concentration of the stock drug solutions was 0.1412 mM. A blank solution with the same volume of buffer and water was prepared for 100 ml. Different volumes of neat pluronic L64, F127, CTAB, SDS, and their mixed micelles were added to a fixed quantity of drug ACF (0.569 µM). They were sonicated and kept at room temperature for stabilization before taking the readings. The water bath sonicator was operated for 20 min at room temperature.

UV-spectrophotometry
UV Spectroscopic measurements were carried out for ACF and ACF with pure and mixed micellar systems. The instrument used was Shimadzu UV-2600K with a 1 cm quartz cell. From the stock solution of ACF, suitable dilution was done and a calibration curve was prepared. It is presented in Fig. S 1 .

Fourier transform infrared spectrum
The FTIR spectra of drug ACF in the absence and presence of the pure and mixed micelles were recorded at 298 K by using a Carry-630 FTIR, Agilent technologies. The range chosen was 400 to 4000 cm −1 . For each reading 1:1 proportion of drug ACF with surfactants (single/mixed) was added. The paste form was ground well in a mortar and infra-red measurements were taken.

Conductivity studies
The specific conductance of the ionic surfactants viz. CTAB and SDS in presence of pluronic L64 and drug ACF were measured using a PICCO-180 conductivity meter with a platinum electrode dipped in solution. All the measurements were carried out at 300 K. Calibration with standard potassium chloride solution was done before starting the experiment. Different concentrations of stock surfactant solution, either single or mixed micelles were measured in a beaker, and measurements were taken with and without ACF. All the chosen concentrations were around the CMC of the surfactants. The specific conductivity values were plotted against the concentration of the ionic surfactants. From the inflection point, the CMC of pure ionic surfactant and mixture containing ionic surfactant + nonionic surfactant L64 + ACF were determined. In both cases, there was a reduction in CMC. From the plots, the degree of dissociation, β was determined. β is related to α through formula, α is calculated by using the formula, where S 1 and S 2 represent the slope of post and pre micellar concentration respectively. From β, the ΔG, ΔH, ΔS, and equilibrium constant was calculated using the formula, where T is temperature, X CMC is CMC in mole fraction and R is the gas constant = 8.314 J/mol K From the above equation plotting to log X cmc vs T gives a straight line with a slope (logX cmc )/∂T, the entropy was obtained by the formula, And, the equilibrium constant K was calculated using the equation where ΔG is Gibbs free energy, R is gas constant and T is temperature.

DLS studies
The changes in the micellar size due to the drug aceclofenac in the mixed micellar system were measured by dynamic light scattering using a zeta nanosizer, ZS (Marvern. Panalytical, UK.). All the readings were taken at 25.0 ± 0.1 °C. The light source used was a 4 mW He-Ne laser (633 nm). The scattering angle of 173 0 was maintained uniformly for the samples.

SEM studies
SEM was used to study topographical and morphological information about the sample surface using JSM-6610 LV instrument. The observation maximum of the sample was up to 5 µm in diameter. At 30 kV, the high-resolution measurement was very clear with fine structures. The SEM images of ACF in two mixed micellar systems viz. L64-CTAB (non-ionic-cationic) and L64-SDS (non-ionic-anionic) were taken. Each sample from the stock solution was taken (2 ml volumes) and dried on a glass piece. After drying, the solids left behind were subjected to SEM analysis. Three samples viz., 1 ml L64 + ACF, 1 ml L64 + 1 ml CTAB + ACF, and 1 ml L64 + 1 ml SDS + ACF were used for studies.

UV visible studies
During drug surfactant interaction, there is a change in hydrophobicity and complex formation which are detected by UV-visible spectroscopy. A Calibration of aceclofenac in an aqueous solution was constructed which is shown in Supplementary Section S 1 . The structures of pluronic L64, F127, CTAB, and SDS have also been shown in S 2 of the Supplementary Section. ACF in aqueous solution displayed the λ max at 274 nm [11,12]. Adding pluronic L64 and F127 to an aqueous solution of aceclofenac did not shift λ max Fig. 1a, b. There was only a linear increase in absorbance with an enhancement of the surfactant concentration. Hence it indicates the interaction and capture of more drugs in the micelles of both the surfactants. A binary mixture of L64 and F127 with drug ACF also displayed increased absorbance linearly with rising in the concentration of surfactant solutions as shown in Fig. 1c. Similar findings have been reported earlier [13][14][15] In Fig. 1d a comparison of the interaction of ACF with L64, F127, and a binary mixture of L64 + F127 has been displayed. It clearly shows that the mixture has higher encapsulation efficiency than the respective single micellar systems. In two more binary systems comprising nonionic L64 with cationic CTAB and anionic SDS systems, the aceclofenac interaction was tried. Figure 2a presents the change of absorbance values of ACF on L64 + CTAB systems. Here, the λ max of ACF was observed to shift from 274 to 280 nm and at 267 nm there is an isosbestic point. The redshift by 6 nm and the isosbestic at blue shift (7 nm) can be accounted for the substitution taking place due to complexation between the mixed micelle of L64 + CTAB with ACF. With the increase in the concentration of the mixed micelle of L64 + CTAB, the constant quantity of drug displayed a linear increase in the absorbance value. This has been shown in Fig. 2a.
In another set of binary ionic mixtures comprising L64 and SDS, the interaction of ACF was noticed. In this case, there was no shift of spectra, only the absorbance increased linearly with an increase in mixed micellar concentration. The first spectrum of Fig. 2b is for neat ACF. The second and third stand for ACF + L64 and ACF + SDS respectively. Both have higher absorbance than the ACF spectrum. This means that ACF has better solubility in L64 and SDS systems than in an aqueous medium. The fourth spectrum represents ACF + L64 + SDS showing higher absorbance than ACF + L64 and ACF + SDS. It can be inferred that L64 + SDS is a successful mixed micellar system for ACF showing more solubilization than its single micellar systems of L64 and SDS.
To have a comparison among the L64-F127 (nonionic-nonionic), L64-CTAB (nonionic-cationic), and L64-SDS (nonionic-anionic), three mixed micellar systems as far as interaction with ACF is concerned, a plot of absorbance was taken. Figure 2c shows that both the ionic-nonionics are more effective compared to the nonionic-nonionic combinations. Also, anionic mixed micelles have better encapsulation capacity than cationic mixed micelles. The interaction order with ACF is thus L64-F127 < L64-CTAB < L64-SDS.

FTIR spectra
The structural changes of ACF due to the interaction of ionic mixed micelles and their single micelles were observed by FTIR studies. IR studies of surfactants have been reported before [16][17][18]. The spectra of ACF and combinations of three neat surfactants L64, CTAB, and SDS were taken. Next, two ionic mixed micellar systems, viz. L64 + CTAB and L64 + SDS were taken for drug interaction. The spectra of 6 samples have been displayed in Fig. 3. The ACF molecule displayed strong signals at 3317 cm −1 , 1713 cm −1 , 1138 cm −1 and 746 cm −1 . The 3317 cm −1 is assigned for NH stretching vibrations 1713 cm −1 for C=O stretching vibration, 1138 cm −1 for C-O stretching vibration, and 746 cm −1 for the Cl − present in the molecule. Besides, there are weaker signals at 1342 cm −1 , and 1280 cm −1 which stand for C-O bonding, and at 1576 cm −1 and 1505 cm −1 for benzene rings present. On addition of pluronic L64 to ACF, there are prominent signals at 2888 cm −1 , 1654 cm −1 , 1092 cm −1 , 770 cm −1 . The 1654 cm −1 is a shift of the 1713 cm −1 of ACF spectrum. This shows that there is a complex formation between ACF and pluronic L64 at 2888 cm −1 , the COOH site, and 1654 cm −1 at C=O of the ACF molecule. Additionally, there is bonding at 1449 cm −1 . The sharp signal at 1092 cm −1 point at shifting of 1138 cm −1 C-O stretching to a lower wavenumber, hence a short and strong bond formation in the complex is indicated.

Conductivity measurements
The conductance measurements give an idea about the behaviour of the ionic mixed micelles in presence of ACF. The conductance of a few surfactants in an aqueous medium has been reported before [19,20].

L64 + CTAB + ACF system
The ternary system of nonionic L64, cationic CTAB comprising the ionic binary mixture was observed for its interaction with ACF. Pure CTAB was observed to have CMC at 0.73 mM. The ternary solution showed the CMC at 0.5 mM.
Hence, there was a lowering of CMC by 0.23 mM. This has been shown in Fig. 4. The ternary solution displayed a linear increase in conductance with an increase in the concentration of ionic surfactants before and after CMC. The change was linear but the values did not rise very high. The change in conductance was from 0.35 to 0.73 mS cm −1 . The pre and post-micellar slopes S 1 and S 2 were calculated to get β, the counter ion binding constant [21]. The β values for the CTAB group were calculated to be 0.666. Using β, the ΔG, ΔH, and ΔS values were calculated to be − 25.4 kJ/mol, − 7.6 kJ/mol, and − 17.7 kJ/mol respectively.
The −ve values of ΔG predict that there is the spontaneity of reaction to form the complex with the ternary mixture of CTAB, L64, and ACF. The equilibrium constant also was calculated to be 1.018 dm 3 /mol. These values have been shown in Table 1.

L64 + SDS + ACF systems
The binary anionic system was seen to interact with ACF remarkably compared to the cationic one.
The results have been shown in Fig. 5 Here the change in conductance (increase) was linear. The range was from 0.42 to 2.47 mS cm −1 . The CMC of SDS is 0.86 mM and that of the ternary solution was 0.43 mM. Hence, there was a lowering of 0.43 mM. Using the pre and post micellar slopes, the β, ΔG, ΔH, and ΔS was calculated. The value of β was 0.533, a value lower than the CTAB mixture. This is because SDS tends to reunite after dissociation with S + and DS −1 ions [22]. The ΔG value was − 27.8 kJ/mol, a higher negative value predicting that the reaction is spontaneous and a more stable product is formed compared to the CTAB group. The ΔH and ΔS values, also hint at stability. Overall, it can be inferred that the SDS mixed micellar system has a better stronger binding to form a complex of higher molecular weight compared to CTAB mixed micelle as far as the interaction with ACF is concerned.

SEM analysis
The incorporation of the drug ACF in the mixed micelle of pluronic L64 + CTAB and L64 + SDS were done and SEM images were taken. The images are shown in Figs. 6 and 7.   Figure 6a shows an image of a drug incorporated in L64. The surface is somewhat uniform at 50 µm resolution. In Fig. 6b L64 + CTAB + ACF has been displayed wherein 10 µm and high resolutions (50 µm), there are flowery-shaped bigger crystals. This may be because of the high molecular weight of CTAB (364.4 g/mol). On the contrary, the mixed micelle containing SDS (molecular weight 288 g/mol) shows smaller-sized flowery crystallike shapes as displayed in Fig. 7c. Thus, the surface morphology of the ACF captured in two different types of mixed micelles shows different types of structures. The

DLS studies
The changes in the structure of the pure and mixed micellar due to the incorporation of ACF were observed from the particle size measurement by DLS studies. The results are shown in Table 2. Pluronic micelles are known to display changes in size due to micellar encapsulation [24,25]. As observed, the diameter of pure L64 micelle was 98 nm. This value is agreeable with literature values. The addition of the drug ACF changes the size to 168 nm. This is about a 70 nm increase. This is due to the drug encapsulation in the single micellar cavity of pluronic L64. In the next step, ACF was added to the mixed micelle of L64 + CTAB, increasing the diameter to 329 nm. Its incorporation in L64 + SDS mixed micelle displayed a diameter of 291 nm. The enhanced micellar size observed in these mixed micelle + ACF combinations may be due to either the ACF molecules incorporated in the micellar cavity, or they may have intercalated within the self-assembly cover. So, a larger micellar size may be a result either of (1) a greater number of ACF molecules, or (2) a greater number of surfactant molecules. Since the constant volume and concentration of the surfactant pluronic L64 are used in all four cases, the only possibility is more ACF molecules are accommodated in the mixed micelle. Hence, one can infer that there is more encapsulation of the drug in both the mixed micellar cavities compared to the single micelle of L64. The higher values of CTAB + L64 may be due to the higher molecular weight of CTAB compared to that of SDS. Overall, this study also infers that the ionic binding in mixed micelles is more effective than a single L64 for ACF.

Conclusion
Three mixed micellar systems were explored to observe the interaction of ACF. There was enhanced solubilization of ACF in all the three mixed micellar systems compared to their single micellar ones. The studies also suggested that the ionic mixed micelles undergo more effective interactions with ACF than the non-ionic mixed micelle. Further, the anionic mixed pair has a better effect than the cationic pair. From the conductivity studies, the counter ion binding constant and free energy of micellization infers that the ionic mixed micelle interactions are thermodynamically feasible and higher negative value of anionic mixed micelle (− 27.8 kJ/mol) compared to cationic mixed micelle (− 25.4 kJ/mol) prove that the former is more stable than the latter. The above study infers that a surfactant can be tuned through mixed micellar formulation to have better solubilization of ACF than single micellar solutions. Additionally, an anionic mixed micellar system can be preferred to a cationic mixture for ACF. These findings can be extended to other hydrophobic drug systems.
Funding The authors have not taken any monetary help from any sources.
Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Conflict of interest
The authors declare that they have no conflict of interest.
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