HPLC Assay for Simultaneous Determination of LTZ and CXB

Supplementary Material Experimental

HPLC assay for simultaneous determination of LTZ and CXB An HPLC method was developed and validated for simultaneous determination of LTZ and CXB. An Agilent 1260 Infinity HPLC system equipped with a quaternary pump, an autosampler, DAD (Diode Array Detector), and an Agilent Chemstation data processing system was used for the analysis. The HPLC analysis was carried out with an Agilent Zorbax C18 reversed-phase column (250×4.6 mm, 5 µm particle size) maintained at room temperature. For chromatographic elution, the flow rate was 1.25 ml/min over the entire separation and the injection volume was 20 µL. A step gradient method was utilized for elution in which the mobile phase consisted initially of 60% methanol and 40% water for the first 5 min, followed by 90% methanol and 10% water for the next 5 min. Total run time was 10 min and LTZ was detected at 239 nm, while CXB was detected at 254 nm.

Solubility study of LTZ and CXB in oils Solubility study of LTZ in 5 different oils was investigated to select the optimum oil for the preparation of drug-loaded oily-core NCs. Excess LTZ powder was added to screw-capped test tubes containing 1 ml of different oils (Capryol 90, Capryol PGMC, Lauroglycol 90, labrafac 1349 lipophile and labrafac PG) and then test tubes were shaken in a thermostatically controlled water bath (Kottermann, type 3047, Hanigsen, Germany) at 37±0.5°C for 24 hr. Samples were then centrifuged at 3000 rpm for 5 min. Aliquots from the samples were suitably diluted with absolute ethanol and analyzed for LTZ by HPLC. The solubility of CXB in Lauroglycol 90 was performed as described above. The concentration of CXB was measured by HPLC. All solubility measurements were performed in triplicate.

Preparation of LTZ-loaded PRM-NCs Two different procedures were used for preparation of LTZ-loaded PRM-NCs; the first procedure, called one-stage procedure involves adding 0.5 ml of oil containing 10 mg LTZ to an organic phase consisting of lipoid S75 (50 mg) dissolved in 10 ml ethanol. This organic phase was added to an aqueous phase (50 ml) that contains 50 mg PRM and Tween ®80 (0.2% w/v) under magnetic stirring at 200 rpm for 30 min. The above solution was rota-evaporated at 45°C and 50 rpm for 10 min until a volume of 10 ml. The second procedure, called two-stage procedure consists of adding 0.5 ml of oil containing 10 mg LTZ to an organic phase consisting of lipoid S75 (50 mg) dissolved in 10 ml of ethanol. This organic phase was added to an aqueous phase (50 ml) containing Tween ®80 (0.2% w/v). The formation of the nanoemulsion was instantaneous, which was evident due to the milky appearance of the mixture. The above solution was rota-evaporated at 45°C and 50 rpm for 10 min until a volume of 10 ml, and then incubated with an aqueous solution of PRM (50 mg) in a volume ratio of 1:2.5 (PRM solution:nanoemulsion) under magnetic stirring at 200 rpm for 30 min.

Results and discussion LTZ-loaded PRM-NCs

1 LTZ -loaded PRM-NCs were successfully prepared by simple polymer coating technique which is dependent on O/W spontaneous emulsification process upon mixing of the aqueous phase with a miscible ethanolic oily drug phase. The first approach was the one-stage technique, which allowed the emulsification process to occur simultaneously with the attachment of the cationic outer polymer (PRM) corona to the nanoemulsion surface in a single step (Oyarzun-Ampuero et al., 2013). Solubility of LTZ in five different oils (Capryol 90, Capryol PGMC, Labrafac 1349, Labrafac PG and Lauroglycol 90) was investigated in our study (Figure S1). Maximum LTZ solubility was achieved with Capryol 90 (3.5 mg/mL). Capryol PGMC and Lauroglycol 90 showed also high LTZ solubility (2.5 and 1.6 mg/mL, respectively). Table S1 shows the composition and characteristics of LTZ-loaded PRM-NCs prepared by one-stage procedure using those three oils (F1-F3). To further increase drug loading, the oil solubility of both drugs was enhanced by heating to 50°C during preparation of NCs. LTZ-NCs prepared by Capryol 90 and Capryol PGMC exhibited larger PS > 400 nm and %EE of about 84.5% and PDI (0.453±0.02 and 0.439±0.04 nm, respectively) as compared with LTZ-NCs prepared with Lauroglycol 90 that showed much smaller PS (211.9±6.66 nm), smaller PDI (0.292±0.02) and higher LTZ EE of 91.3%. So, Lauroglycol 90 was chosen as the oily core to prepare drug-loaded PRM-NCs. The nanoemulsion (NE) of F3 exhibited PS of 124.3±5.70 nm, PDI of 0.149±0.01 and zeta potential of -38.1 mV. From results shown in Table 8, it is worth noting that one-stage procedure produced highly negatively charged nanoemulsion (-38.1 mV), this negative charge couldn‘t be reversed by the addition of the positively charged PRM (F3; -23.4 mV). The second approach is the two-stage technique which involved two steps, i.e. an emulsification process resulting in the formation of a negatively charged nanoemulsion followed by coating with the cationic PRM. When the cationic PRM interacts by the ionic interaction with the anionic nanoemulsion, it forms a polymer corona at the oil/aqueous interface thus originating PRM-NCs. the nanoemulsion negative charge which is attributed to the residual free fatty acids present in phospholipids (Oyarzun-Ampuero et al., 2013). Table S2 shows the composition and characteristics of LTZ-loaded PRM-NCs prepared by two-stage procedure. By this procedure and using adequate concentration of PRM (1.25% solution), we prepared NCs with much lower negative charge (Table S2; F4; -6.68 mV) with the formation of homogenous populations of NCs of around 148 nm. As can be noted, decreasing the amount of Lipoid® S75 from 100 to 50 mg could invert the negative charge from (-6.68 mV, F4) to positive charge (+9.57 mV, F5) and reduced the size from 148 to 118 nm. Our results are consistent with Prego et al. (Prego et al., 2006), where an increase in lecithin amount from 40 to 120 mg led to a significant enlargement of the NCs (from 266 to 333 nm), accompanied by a reduction of the surface charge from +34.8 to +28 mV. According to Mosqueira et al. (Mosqueira et al., 2000), the presence of phosphatidic acid, as an impurity in lecithin, is responsible for the negative charge on the particle surface, thus decreasing lecithin amount will decrease the negative charge. Also, we investigated the effect of the type of phospholipid (Lipoid® S 75, S PC3 and S 100). Three PRM-NCs were successfully prepared (Table S2; F5-F7) that exhibited satisfactory PS, PDI, zeta potential and EE. Actually, we selected (F5) which showed the smallest PS (118.2±7.92 nm) and the highest %EE (95.6%), to complete our study. Further increasing the concentration of the cationic PRM solution resulted in larger PS (F8 & F9), as reported by Dey et al. (Dey et al., 2009). Interestingly, increasing oil (Lauroglyco 90) volume from 0.5 to 0.75 and 1 mL, showed a favorable increase in the positive charge (from +9.57 to +26.8 and +15.6

2 mV, respectively) (F5, F10 and F11). Regarding PS, 0.75 mL oil almost did not change the PS, while 1 mL oil caused a marked increase in PS from (118.2±7.92 to 183.3±5.16 nm) (Table S2). Previous studies also showed that larger particles were formed when the oil concentration is increased (Wohlgemuth et al., 2000). Increasing LTZ amount from 10 to 25 mg caused a significant increase in PS (122.4±0.71 to 173.7±1.99 nm) and decrease in zeta potential (+26.8 to +13.4 mV) (Table S2; F10 and F12), whereas 50 mg LTZ could not be dissolved in 0.75 mL Lauroglycol oil. These results were parallel to that of Awotwe-Otoo et al. (Awotwe-Otoo et al., 2012), where an increase in the amounts of drug and polymer resulted in a corresponding increase in the particle size. Concerning reduction in zeta potential value, it is directly proportional to electrophoretic mobility (ratio of velocity of migration over potential gradient). The higher the average NP size, the slower the velocity of migration of charged particles in a known applied electric potential resulting in decreased zeta potential value compared to smaller size NPs, which has higher velocity of migration and higher zeta potential value (Egorova, 1994). Overall, the optimal factors were selected on the basis of smaller PS, higher positive zeta potential and good %EE of the prepared NCs. Therefore, F10 was selected, which exhibited a PS of 122.4±0.71 nm, zeta potential of +26.8 mV and LTZ-EE of 84.7%, to complete our study and add our second drug CXB to formulate the combined LTZ-CXB-loaded PRM-NCs.

Combined LTZ-CXB-loaded PRM-NCs

LTZ and CXB-loaded PRM-NCs were successfully prepared by simple polymer coating technique which is dependent on O/W spontaneous emulsification process upon mixing of the aqueous phase with a miscible ethanolic oily drug phase. CXB solubility study in our selected oil, Lauroglycol 90, was performed and found to be 18 mg/mL. Table S3 shows the composition and characteristics of dual LTZ-CXB-loaded PRM-NCs prepared by two-stage procedure. The results showed that addition of 10 mg CXB to LTZ-loaded PRM-NCs to form combined LTZ- CXB-loaded PRM-NCs resulted in an increase in the PS from 122.4 nm (F10, Table S3) to 139.9 nm which further increases to 143.2 nm by increasing amount of CXB added to 20 mg (Table S3; F13 and F14, respectively). In concordance with Zheng et al. (Zheng et al., 2010), increasing CXB from 10 to 20 mg led to an apparent reduction in the encapsulation efficiency of both LTZ (from 95.9 to 83%) and CXB (from 95.6 to 92.7%). The effect of PRM concentration (0.25-2.5% w/v) was investigated (F15-F18). Addition of a low amount of PRM (0.25% w/v) produced NCs with negative zeta potential (-20.5 mV) and large PS (144.3±1.06 nm), by increasing PRM concentration to 0.5% (w/v) an increase in the zeta potential leading to inversion of the negative charge and insignificant change in PS were observed. Using PRM concentration of 1% (w/v) produced better NCs with high positive zeta potential (+19.0 mV), significantly smaller PS (109.7±6.47 nm) and high LTZ and CXB EE (86.36 and 91.24%, respectively). Further increase in PRM concentration showed a marked increase in PS but insignificant change in the positive zeta potential and this was probably due to the fact that the surface of the oily core was saturated in PRM (Fig. S2 and S3). The dependency of the zeta potential with the amount of PRM evidenced the surface localization of PRM molecules and indicated the necessity of using a minimum amount of PRM to produce stable NCs with suitable PS and high positive zeta potential (Bender et al., 2012). Based on these results, a PRM concentration of 1% (w/v) which assures the surface saturation by the polymer was selected to formulate the optimized LTZ-CXB-loaded PRM-NCs formulation.

3 References Awotwe-Otoo, D., Zidan, A.S., Rahman, Z., Habib, M.J., 2012. Evaluation of anticancer drug-loaded nanoparticle characteristics by nondestructive methodologies. AAPS PharmSciTech 13, 611-622. Bender, E.A., Adorne, M.D., Colomé, L.M., Abdalla, D.S., Guterres, S.S., Pohlmann, A.R., 2012. Hemocompatibility of poly (ɛ-caprolactone) lipid-core nanocapsules stabilized with polysorbate 80- lecithin and uncoated or coated with chitosan. Int. J. Pharm. 426, 271-279. Dey, S.K., Mandal, B., Bhowmik, M., Ghosh, L.K., 2009. Development and in vitro evaluation of Letrozole loaded biodegradable nanoparticles for breast cancer therapy. Brazilian Journal of Pharmaceutical Sciences 45, 585-591. Egorova, E.M., 1994. The validity of the Smoluchowski equation in electrophoretic studies of lipid membranes. Electrophoresis 15, 1125-1131. Mosqueira, V.C.F., Legrand, P., Pinto‐Alphandary, H., Puisieux, F., Barratt, G., 2000. Poly (D, L‐lactide) nanocapsules prepared by a solvent displacement process: Influence of the composition on physicochemical and structural properties. J. Pharm. Sci. 89, 614-626. Oyarzun-Ampuero, F.A., Rivera-Rodríguez, G.R., Alonso, M.J., Torres, D., 2013. Hyaluronan nanocapsules as a new vehicle for intracellular drug delivery. Eur. J. Pharm. Sci. 49, 483-490. Prego, C., Fabre, M., Torres, D., Alonso, M., 2006. Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery. Pharm. Res. 23, 549-556. Wohlgemuth, M., Machtle, W., Mayer, C., 2000. Improved preparation and physical studies of polybutylcyanoacrylate nanocapsules. J. Microencapsul. 17, 437-448. Zheng, Y., Yu, B., Weecharangsan, W., Piao, L., Darby, M., Mao, Y., Koynova, R., Yang, X., Li, H., Xu, S., 2010. Transferrin-conjugated lipid-coated PLGA nanoparticles for targeted delivery of aromatase inhibitor 7α-APTADD to breast cancer cells. Int. J. Pharm. 390, 234-241.

Figures

Figure S1. Solubility of LTZ in different oils at 37°C.

Figure S2. The influence of concentration of PRM solution (0.25 – 2.5% w/v) on the particle size of PRM-NCs.

Figure S3. The influence of concentration of PRM solution (0.25 – 2.5% w/v) on the zeta potential of PRM-NCs.

Table S1. Composition and physicochemical characteristics of LTZ-loaded PRM-NCs prepared by one-stage procedure.

4 Formu Oil PS ζ-potential LTZ EE PDI la (0.5 mL) (nm) (mV) (%w/w) Capryol F1 495.8±9.31 0.439±0.04 -29.80 84.64 PGMC F2 Capryol 90 428.2±1.13 0.453±0.02 -25.80 84.53 Lauroglycol F3 211.9±6.66 0.292±0.02 -23.40 91.30 90 * All formulations were prepared with 10 mg LTZ, 100 mg Lipoid S75, 50 mg PRM, 0.5 mL oil, 0.2% w/v Tween 80.

Table S2. Composition and physicochemical characteristics of LTZ-loaded PRM-NCs prepared by two-stage procedure.

LTZ Lecithin LG90 PRM PS ζ-potential LTZ EE No. PDI (mg) (mg) (ml) (%w/v) (nm) (mV) (%w/w)

Lipoid F4 10 0.5 1.25 148.2±3.91 0.204±0.02 -6.68 93.70 S75 (100) Lipoid F5 10 0.5 1.25 118.2±7.92 0.264±0.03 +9.57 95.60 S75 (50) Lipoid F6 10 0.5 1.25 201.5±0.92 0.260±0.001 +9.40 90.40 S100 (50) Lipoid F7 10 0.5 1.25 248.3±5.44 0.300±0.004 +9.55 94.40 SPC3 (50) Lipoid F8 10 0.5 1.875 173.6±3.75 0.219±0.004 +5.40 92.20 S75 (50) Lipoid F9 10 0.5 2.5 184.3±0.14 0.278±0.03 +3.42 83.60 S75 (50) Lipoid F10 10 0.75 1.25 122.4±0.71 0.216±0.001 +26.80 84.70 S75 (50) Lipoid F11 10 1 1.25 183.3±5.16 0.258±0.001 +15.60 85.30 S75 (50) Lipoid F12 25 0.75 1.25 173.7±1.99 0.137±0.01 +13.40 83.58 S75 (50) * LG90: Lauroglycol 90, P188: Poloxamer-188. Table S3. Composition and physicochemical characteristics of LTZ-CXB-loaded PRM-NCs prepared by two-stage procedure.

5 CXB PRM PS ζ-potential LTZ EE CXB EE No. PDI (mg) (% w/v) (nm) (mV) (%w/w) (%w/w)

F13 10 1.25 139.9±5.46 0.208±0.006 +17.29 95.60 95.60

F14 20 1.25 143.2±7.98 0.219±0.01 +18.30 83.00 92.70

F15 20 0.25 144.3±1.06 0.295±0.05 -20.50 86.10 91.30

F16 20 0.5 132.5±5.06 0.249±0.003 +7.16 84.30 92.30

F17 20 1 109.7±6.47 0.313±0.007 +19.00 86.36 91.24

F18 20 2.5 154.6±8.06 0.285±0.002 +18.40 83.20 93.40 * All formulations were prepared with 10 mg LTZ, 0.75 mL Lauroglycol 90, 50 mg Lipoid® S75, 0.2% (w/v) Tween 80, 1:2.5 (v:v) PRM:NE.