Preparation of Nonionic Vesicles Using the Supercritical Carbon Dioxide Reverse Phase Evaporation Method and Analysis of Their Solution Properties

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1 Journal of Oleo Science Copyright 2016 by Japan Oil Chemists Society J-STAGE Advance Publication date : December 11, 2015 doi : /jos.ess15192 Preparation of Nonionic Vesicles Using the Supercritical Carbon Dioxide Reverse Phase Evaporation Method and Analysis of Their Solution Properties Shunsuke Yamaguchi 1, 2, Koji Tsuchiya 3, Kenichi Sakai 2, 3, Masahiko Abe 3 and 2, 3,* Hideki Sakai 1 Cosmos Technical Center Co., Ltd., Hasune, Itabashiku, Tokyo , JAPAN 2 Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba , JAPAN 3 Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba , JAPAN Abstract: We have previously reported a new preparation method for liposomes using supercritical carbon dioxide (scco 2 ) as a solvent, referred to as the supercritical carbon dioxide reverse phase evaporation (scrpe) method. In our previous work, addition of ethanol to scco 2 as a co-solvent was needed, because lipid molecules had to be dissolved in scco 2 to form liposomes. In this new study, niosomes (nonionic surfactant vesicles) were prepared from various nonionic surfactants using the scrpe method. Among the nonionic surfactants tested were polyoxyethylene (6) stearylether (C 18 EO 6 ), polyoxyethylene (5) phytosterolether (BPS-5), polyoxyethylene (6) sorbitan stearylester (TS-106V), and polyoxyethylene (4) sorbitan stearylester (Tween 61). All these surfactants have hydrophilic-lipophilic balance values (HLBs) around 9.5 to 9.9, and they can all form niosomes using the scrpe method even in the absence of ethanol. The high solubility of these surfactants in scco 2 was shown to be an important factor in yielding niosomes without ethanol addition. The niosomes prepared with the scrpe method had higher trapping efficiencies than those prepared using the conventional Bangham method, since the scrpe method gives a large number of unilamellar vesicles while the Bangham method gives multilamellar vesicles. Polyoxyethylene-type nonionic surfactants with HLB values from 9.5 to 9.9 were shown to be optimal for the preparation of niosomes with the scrpe method. Key words: nonionic surfactant, supercritical dioxide reverse phase evaporation method, niosome, liposome 1 INTRODUCTION Liposomes are self-enclosed aggregates with lipid bilayers encapsulating an inner solution phase 1. They have been used as models for biological membranes because their structures and functions are similar to those of biological membranes 2 5. One of the most useful properties of liposomes is their retention of water-soluble substances in the inner aqueous phase and oil-soluble substances in the bilayers 6, 7. Because of this, liposomes are widely used in both drug formulations and cosmetics. Although there are many preparation methods for liposomes 1, 8, 9, most of them including the Bangham method and the reverse phase evaporation method require use of organic solvents, which can be harmful to the human body and/or the environment. As such, when liposomes are used as drug carriers or in cosmetics, organic solvents must be avoided whenever possible. Additionally, liposomes prepared by the Bangham method generally have lower trapping efficiencies for water-soluble drugs than those prepared by the reverse phase evaporation method, as the former yields multilamellar vesicles MLVs while the latter gives large unilamellar vesicles LUVs. Based on this information, we have previously reported a supercritical carbon dioxide reverse phase evaporation scrpe method for the preparation of liposomes in a single step using supercritical CO 2 scco 2 and a small amount of ethanol 10. In this method, scco 2 which can be removed from the system by reducing the pressure was used instead of organic solvents such as chloroform. The liposomes prepared using the scrpe method gave larger * Correspondence to: Hideki Sakai, Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba , JAPAN hisakai@rs.noda.tus.ac.jp Accepted October 9, 2015 (received for review August 7, 2015) Journal of Oleo Science ISSN print / ISSN online 1

2 S. Yamaguchi, K. Tsuchiya and K. Sakai et al. trapping efficiencies for water-soluble substances compared to those prepared with the Bangham method. Niosomes, which are bilayer vesicles consisting of nonionic surfactants, have structures and properties similar to liposomes. They are more suitable than liposomes for industrial applications because of their high chemical stability and low cost. Furthermore, there are a large number of nonionic surfactants available for the design of niosomes, because there are many possible combinations of hydrophilic and lipophilic groups. We have recently succeeded in the preparation of niosomes using the scrpe method in the absence of ethanol 11. However, niosome formation with the scrpe method has so far only been confirmed for polyoxyethylene alkyl ethers. If more molecular structures and preparation conditions suitable for niosome preparation can be found, this will provide valuable information to further the application of niosomes to drug delivery systems and cosmetics. In this study, the scrpe method was used to prepare niosomes with various types of nonionic surfactant. The effects of the molecular structure of the nonionic surfactants on niosome formation and physicochemical properties were examined via trapping efficiency measurements, transmission electron microscopy TEM, and dynamic light scattering DLS. 2 EXPERIMENTAL 2.1 Materials The polyoxyethylene alkyl ethers C n EO m, n number of carbon in the hydrophobic group, m number of oxyethylenes, polyoxyethylene sorbitan alkyl esters TS-106V and Tween 61, and polyoxyethylene phytosterol ethers BPS-5, BPS-10 and BPS20 listed in Table 1 were used as polyoxyethylene POE -type nonionic surfactants. The sorbitan alkyl esters SM and SS and polyglycerol fatty acid esters TG1S, HG1S, and DG2S listed in Table 2 were used as nonionic surfactants without POE as the hydrophilic group. All surfactants except Tween 61 were provided by Table 1 Nonionic surfactants with polyoxyethylene as hydrophilic groups used in this study. Chemical structure of nonioinic surfactants Polyoxyethylene alkyl ether HLB* Polyoxyethylene (4) laurylether (C 12 EO 4 ) 9.7 Polyoxyethylene (5) laurylether (C 12 EO 5 ) 10.8 Polyoxyethylene (7) laurylether (C 12 EO 7 ) 11.7 Polyoxyethylene (2) stearylether (C 18 EO 2 ) 4.9 Polyoxyethylene (4) stearylether (C 18 EO 4 ) 7.9 Polyoxyethylene (6) stearylether (C 18 EO 6 ) 9.9 Polyoxyethylene sorbitan fatty acid ester Polyoxyethlyene (6) sorbitan monostearate (TS-106V) 9.5 Polyoxyethlyene (4) sorbitan monostearate (Tween 61) 9.6 Polyoxyethylene phytosterol ether Polyoxyethylene (5) phytosterol (BPS-5) 9.5 Polyoxyethylene (10) phytosterol (BPS-10) 12.5 Polyoxyethylene (20) phytosterol (BPS-20) 15.5 * HLB values were given by suppliers. 2

3 Preparation of Nonionic Vesicles Using the Supercritical Carbon Dioxide Reverse Phase Evaporation Method and Analysis of Their Solution Properties Table 2 Nonionic surfactants without polyoxyethylene as hydrophilic groups used in this study. Chemical structure of nonioinic surfactants Sorbitan fatty acid ester HLB* Sorbitan monomyristate (SM) 7.7 Sorbitan monostearate (SS) 4.7 Polyglycerol fatty acid ester R = H or stearyl Tetraglycerol monostearate (TG1S) 6.0 Hexaglycerol monostearate (HG1S) 9.0 Decaglycerol distearate (DG2S) 9.5 * HLB values were given by suppliers. Nikko Chemicals Co., and Tween 61 was obtained from Sigma Chemical Co. These materials were used without further purification. 2.2 Methods scrpe method Figure 1 shows a schematic illustration of the experimental apparatus used for the scrpe method 10. After a nonionic surfactant was sealed in the cell, CO 2 was introduced. The cell temperature was then raised to 40, while the pressure was kept at bar. After waiting a few minutes to allow equilibration, a 0.2 M aqueous solution of glucose a water-soluble model drug substance was introduced at a rate of 0.1 ml/min using a HPLC pump until total volume of 5 ml and the surfactant concentration of 10 mm. The pressure was then reduced by CO 2 ejection and yielding suspensions. The interior of the cell was stirred with a magnetic stirring bar during the preparation process Bangham method Niosomes were also prepared with the conventional Bangham method in order to compare the results with those from the scrpe method. A nonionic surfactant was dissolved in chloroform in a test tube. The solvent was then removed by blowing nitrogen gas into the test tube, and the residual material was further dried overnight at room temperature under vacuum to give a thin surfactant film on the wall of the test tube. An aqueous solution of glucose 0.2 M was added, and the sample was warmed to 60 for 10 min. The test tube was then vigorously shaken with a vortex mixer to yield multi-lamellar niosomes Evaluation of niosome suspensions To determine the glucose trapping efficiency of the niosomes, the glucose-loaded niosome suspension was dialyzed against water using a cellophane tube Viscase Scales Co. to remove unentrapped glucose. The niosomes inside the tube were then destroyed by the addition of ethanol. The amount of glucose in the solution was determined by the mutarotase glucose oxidase method 12, 13 using a spectrophotometer UV-260, Shimadzu Co.. The hydrodynamic diameter of the niosomes was estimated using a NICOMP 380ZLS particle size analyzer equipped with a 5 mw He-Ne laser at a constant detector angle of 90. The obtained scattering data were fitted using a number-weighted NICOMP-mode analysis to estimate the diffusion coefficient of the surfactant assemblies in the solution. The hydrodynamic diameter was obtained from the diffusion coefficient using the Stokes-Einstein equation. The formation of niosomes was confirmed by cryogenic transmission electron microscopy cryo-tem. A small amount 3-5 μl of sample solution was placed on the surface of a TEM copper grid covered by a holey carbon film, which was held by a pair of self-locking tweezers mounted on a spring-loaded shaft with a cryo-preparation system LEICA EM CPC, LEICA microsystems. The sample drop was blotted with filter paper to form a thin liquid film on the grid thickness 300 nm, and immediately plunged into liquid ethane cooled by liquid nitrogen 170. The grid was transferred onto the tip of a cryospecimen holder CT-3500, Oxford Instruments under liquid nitrogen. Specimens were kept at temperatures below 170 and imaged using a transmission electron microscope H-7650, Hitachi Science Systems, Ltd. at an accelerating voltage of 120 kv under a low electron dose. Fig. 1 Apparatus for preparation of niosomes with the scrpe method. 3

4 S. Yamaguchi, K. Tsuchiya and K. Sakai et al. 3 RESULTS AND DISCUSSION 3.1 Preparation of niosomes using POE-type noninonic surfactants Hal et al. reported that niosomes were formed by nonionic surfactants with hydrophilic-lipophilic balance HLB values between 7.5 and In general, HLB values are simply derived from the molecular formula, and do not reflect the conformation and packing of the surfactant molecules in self-assemblies. In this study, niosomes using POE-type nonionic surfactants were first prepared by the conventional Bangham method as a reference. The formation of niosomes was confirmed through the existence of lamellar structures, which were observed by polarized optical microscopy POM, IMT-2, OLYMPUS Co., and the existence of the inner aqueous phase was evaluated by trapping efficiency measurements. The Maltese cross optical pattern, which is characteristic of a lamellar structure, was observed by POM for C 12 EO 4, C 18 EO 4, C 18 EO 6, TS- 106V, Tween 61, and BPS-5. Table 3 shows the results of the trapping efficiency measurements at a concentration of 10 mm, which confirm the existence of an inner aqueous phase for these six nonionic surfactant systems. These results suggested the formation of niosomes using the Bangham method. The HLB values of these nonionic surfactants range from 7.9 to 9.9, which are similar to those reported by Hal 14. The scrpe method was then used to attempt to form niosomes with the nonionic surfactants which gave niosomes by the Bangham method. Of these surfactants, C 18 EO 6, TS-106V, Tween 61, and BPS-5 were able to form niosomes using the scrpe method in the absence of ethanol, as proven by the existence of the aqueous inner phase confirmed by the trapping efficiency measurements Table 3. The niosomes prepared by the scrpe method had higher trapping efficiencies than those prepared by the Bangham method. Although niosomes were formed by C 12 EO 4 and C 18 EO 4 using the Bangham method, phase separation immediately occurred in suspensions prepared by the scrpe method. The trapping efficiencies for these nonionic surfactant suspensions were thus almost zero. Consequently, the HLBs of the nonionic surfactants which yielded niosomes using the scrpe method without ethanol ranged from 9.5 to 9.9. The formation of niosomes by C 18 EO 6, TS-106V, Tween 61, and BPS-5 using the scrpe method without ethanol was probably caused by the higher solubility of these surfactants in scco Preparation of niosomes using non-poe-type nonionic surfactants Niosomes of nonionic surfactants without POE as a hydrophilic group were prepared using the Bangham method. Table 4 shows their trapping efficiency results. The formation of niosomes by the Bangham method was confirmed for TG1S, HG1S, and DG2S. The preparation of niosomes using the scrpe method was also attempted for these surfactants. However, niosome formation was not confirmed, probably because of the low solubility of these surfactants in scco 2 due to the lack of hydrophilic POE groups. In the scrpe method, water/scco 2 emulsions are formed by the dissolution of the nonionic surfactants in scco 2. After removal of CO 2 from the scco 2 /water emulsion, LUVs are formed. Thus, the solubility of the surfactants in scco 2 would be an important factor for preparation of niosomes by the scrpe method. When the solubility of polyglycerol fatty acid esters in scco 2 was monitored using a high pressure cell with a quartz window, they were found to be insoluble in scco 2, in contrast to the POE-type surfactants. We are currently Table 3 Trapping efficiency of molecular assemblies formed by nonionic surfactants with polyoxyethylene as hydrophilic groups. Nonionic surfactants HLB with Bangham method with scrpe method C 12 EO % 0 % C 12 EO % C 12 EO % C 18 EO % C 18 EO % 0 % C 18 EO % 4.8% TS-106V % 6.0% Tween % 6.0% BPS % 7.2% BPS % BPS % 4

5 Preparation of Nonionic Vesicles Using the Supercritical Carbon Dioxide Reverse Phase Evaporation Method and Analysis of Their Solution Properties Table 4 Trapping efficiency of molecular assemblies formed by nonionic surfactants without polyoxyethylene as hydrophilic groups. Nonionic surfactants HLB with Bangham method with scrpe method SM % SS % TG1S % 0% HG1S % 0% DG2S % 0% investigating the preparation of niosomes using polyglycerol fatty acid ester surfactants by the scrpe method with the addition of a small amount of ethanol. These results will be reported separately at a later date. 3.3 Physicochemical properties of niosomes prepared using the scrpe method Niosomes formed by nonionic surfactants C 18 EO 6, TS106V, Tween 61, and BPS-5 were further investigated using dispersion stability and trapping efficiency measurements. Figure 2 shows photographs of the niosome suspensions after different standing times. Niosomes prepared from C 18 EO 6 and Tween 61 gradually separated into two phases, while BPS-5 precipitated after 3 days. On the other hand, TS-106V niosomes showed good dispersion stability for over 1 month. Figure 3 shows changes in particle size over time of niosomes prepared by the scrpe method. The niosomes prepared using polyoxyethylene sorbitan alkyl esters TS-106V and Tween 61 kept a constant particle size nm for more than four weeks, while those prepared with other surfactants gradually increased to more than 1000 nm. This result suggests that the sorbitan structure leads to high dispersion stability in niosomes. Figure 4 shows a cryo-tem micrograph of TS-106V niosomes prepared by the scrpe method. The formation of LUVs with sizes of around 400 nm was confirmed. Thus, the high trapping efficiency of niosomes prepared by the scrpe method without co-solvents is probably caused by the formation of LUVs, while multilamellar vesicles MLVs are formed by the Bangham method. Fig. 2 Visual appearance of niosome suspensions (C 18 EO 6, TS106V, Tween61 and BPS-5) prepared with the scrpe method as a function of standing time. 4 Conclusions In this study, we attempted to prepare niosomes using various nonionic surfactants using the scrpe method without ethanol as a co-solvent to clarify important factors in their formation. For POE-type nonionic surfactants, C 18 EO 6, TS-106V, Tween 61, and BPS-5 successfully formed niosomes by the scrpe method without ethanol. These POE-type nonionic surfactants have HLB values ranging from 9.5 to 9.9, which is clearly an important factor for the preparation of niosomes using the scrpe method without ethanol. Niosomes prepared using this method had higher trapping efficiencies than those prepared by the conventional Bangham method because of the formation of LUVs. On the other hand, when nonionic surfactants without POE as a hydrophilic group were used, niosomes could not be formed by the scrpe method - even for DG2S, which has a HLV of 9.5. This was probably caused by the poor solubility 5

6 S. Yamaguchi, K. Tsuchiya and K. Sakai et al. Fig. 3 Time course change in particle size of niosomes prepared with the scrpe method. Fig. 4 Cryo-TEM image of TS-106V niosome prepared with the scrpe method. of the non-poe-type surfactants in scco 2. Thus, the high solubility of nonionic surfactants in scco 2 which can be achieved by using POE-type nonionic surfactants is another important factor for the preparation of niosomes using the co-solvent-free scrpe method. These findings provide useful information which could be used for the preparation of niosomes using other nonionic surfactants by this environment-friendly method. References 1 Bangham, A. D.; Standish, M. M.; Watkins, J. C. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13, Balon, K.; Riebesehl, B. U.; Mueller, B. W. Determination of liposome partitioning of ionizable drugs by titration. J. Pharm. Sci. 88, Colic, M.; Morse, D. The elusive mechanism of the magnetic memory of water, Colloids Surf. A 154, Montero, M. T.; Hernandez-Borrell, J.; Keough, K. M. W. Fluoroquinolone-biomembrane interactions: monolayer and caloriemtric studies. Langmuir 14, Komatsu, H.; Okada, S., Increased permeability of phase-separated liposomal membranes with mixtures of ethanol-induced interdigitated and non-interdigitated structures, Biochim. Biophys. Acta 1237, Kaneda Y. Virosomes: evolution of the liposome as a targeted drug delivery system, Adv. Drug Delivery Rev. 43, Marjan, J.; Xie, Z.; Devine, D. V. Liposome-induced activation of the classical complement pathway does not require immunoglobulin. Biochim. Biophys. Acta 1192, Batzru S.; Korn, E. D. Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 298, Szoka, F. Jr.; Papahadjopoulos, D. Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl. Acad. Sci. U. S. A. 75, Otake, K.; Imura, T.; Sakai, H.; Abe, M. Development of a New Preparation Method of Liposomes Using Supercritical Carbon Dioxide. Langmuir 17, Ri, K.; Yamaguchi, S.; Wongtrakul, P.; Hashimoto, S.; Otake, K.; Ohkubo, T.; Sakai, H.; Abe, M. Preparation and Characterization of Nonionic Surfactant Vesicles Using Supercritical Carbon Dioxide. Material Technology 23, Miwa, I.; Okuda, J.; Maeda, K.; Okuda, G. Mutarotase effect on colorimetric determination of blood glucose with -D-glucose oxidase. Clin. Chim. Acta 37, Okuda, J.; Miwa, I. Enzymatic micro-determination of D-glucose and its anomer. Protein, Nucleic Acid, Enzyme 17, van Hal, D. A.; Bouwstra, J. A.; van Rensen, A.; Jeremiasse, E.; de Vringer, T.; Junginger, H. E. Preparation and Characterization of Nonionic Surfactant Vesicles. J. Coll. Interf. Sci. 178,