Preparation of Liposomes from Soy Lecithin Using Liquefied Dimethyl

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Article Preparation of Liposomes from Soy Lecithin Using Liqueﬁed Dimethyl Ether

Hideki Kanda * , Tsubasa Katsube, Wahyudiono and Motonobu Goto

Department of Materials Process Engineering, Nagoya University, Furocho, Chikusa, Nagoya 464-8603, Japan; [email protected] (T.K.); [email protected] (W.); [email protected] (M.G.) * Correspondence: [email protected]

Abstract: We investigated a method to prepare liposomes; soy lecithin was dissolved in liquefied dimethyl ether (DME) at 0.56 MPa, which was then injected into warm water. Liposomes can be successfully prepared at warm water temperatures above 45 ◦C. The transmission electron microscopy (TEM) images of the obtained liposomes, size distribution, ζ-potential measurements by dynamic light scattering and the amount of residual medium were compared by gas chromatography using the conventional medium, diethyl ether. The size of the obtained liposomes was approximately 60–300 nm and the ζ-potential was approximately −57 mV, which was almost the same as that of the conventional medium. Additionally, for the conventional media, a large amount remained in the liposome dispersion even after removal by depressurization and dialysis membrane treatment; however, liquefied DME, owing to its considerably low boiling point, was completely removed by depressurization. Liquefied DME is a very attractive medium for the preparation of liposomes because it does not have the toxicity and residue problems of conventional solvents or the hazards of ethanol addition and high pressure of supercritical carbon dioxide; it is also environmentally friendly. Citation: Kanda, H.; Katsube, T.; Wahyudiono; Goto, M. Preparation of Keywords: subcritical fluid; vesicle; phospholipid; lipid bilayer Liposomes from Soy Lecithin Using Liquefied Dimethyl Ether. Foods 2021, 10, 1789. https://doi.org/10.3390/ foods10081789 1. Introduction

Academic Editors: Branimir Pavlic Liposomes are spherical vesicles with one or more phospholipid bilayers that surround and Danijela BursaćKovaˇcević an aqueous compartment. Liposomes can encapsulate aqueous hydrophilic or hydrophobic substances within the phospholipid bilayer and are often classified as small unilamellar Received: 10 June 2021 vesicles (SUVs), large unilamellar vesicles (LUVs), or multilamellar vesicles (MLVs), based Accepted: 31 July 2021 on their size and the number of phospholipid bilayers [1]. SUVs and LUVs have diameters Published: 2 August 2021 in the range of 20–100 and 100–1000 nm, respectively [2], and are often used as biomaterial carriers [3]. Liposomes are also tools for the oral delivery of nutraceuticals because of their Publisher’s Note: MDPI stays neutral unique properties. Ordinary tablets and capsules have low absorption and bioavailability with regard to jurisdictional claims in properties; thus, the encapsulating fat-soluble and water-soluble nutrients in liposomes are published maps and institutional affil- investigated for the ability to prevent their destruction in the digestive system and facilitate iations. their delivery to cells and tissues. Liposomes can contain a wide variety of hydrophilic and hydrophobic diagnostic and therapeutic agents, providing a relatively large drug payload per particle and protecting the encapsulated drug from metabolic processes [4–6]. The conventional methods of preparing liposomes require toxic organic solvents, Copyright: © 2021 by the authors. complex multistage processes and high energy costs and the resulting liposomes usually Licensee MDPI, Basel, Switzerland. show poor stability. In previous studies, ethanol and diethyl ether were used as organic This article is an open access article solvent injection methods. In the ethanol injection method, phospholipids were dissolved distributed under the terms and in ethanol and rapidly injected into water to produce liposomes. Liposomes with diameters conditions of the Creative Commons ranging from 30 to 110 nm were obtained using this method. One problem is that ethanol Attribution (CC BY) license (https:// mixes azeotropically with water, making it difficult to completely remove ethanol from the creativecommons.org/licenses/by/ liposome dispersion [7,8]. Moreover, the removal of ethanol from an aqueous liposome 4.0/).

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solution requires the distillation of ethanol and water by heating; this results in the destruction of liposomes and encapsulated biomaterials. Additionally, ethanol is not suitable in various religions and carries risks that prevent its use. In the ether injection method, phospholipids were dissolved in diethyl ether and the mixture was injected into water. When the water was heated to 55–65 ◦C, the diethyl ether evaporated and liposomes were formed. The diameter of the liposomes prepared by this method was approximately 70–190 nm [9]. The diethyl ether injection method has been used to encapsulate 5.6-carboxyfluorescein, a hydrophilic drug [10] and a plasmid, a part of the DNA molecule [11]. However, there is concern that the ether may remain in the encapsulated biomaterials. Additionally, diethyl ether must be heated to a considerably high temperature to remove the solvent, making it unsuitable for the encapsulation of heat-sensitive materials. Furthermore, diethyl ether is considerably difficult to handle because it easily produces explosive peroxides and is toxic [1,2]. To solve these problems, the use of organic solvents with low boiling points, such as methyl ethyl ether (boiling point, 10.8 ◦C) and dichlorofluoromethane (boiling point, 8.9 ◦C), has been considered [8]. However, methyl ethyl ether has the same perox- ide problem as diethyl ether and dichlorofluoromethane has been banned because of its ozone depletion and greenhouse effect, which is thousands of times stronger than that of carbon dioxide. To solve this problem, the use of supercritical carbon dioxide (SC-CO2) as a green solvent instead of organic solvents has been proposed. CO2 is not only easy to separate from the product but has also a low critical temperature, providing a mild operating temperature suitable to process and encapsulate thermophilic compounds. Previous studies have shown that various nutraceutical ingredients have been encapsulated in liposomes using SC-CO2 [12–17]. However, because SC-CO2 is non-polar, the solubility of polar phospholipids and water is considerably low; therefore, to incorporate phospholipids and water into SC-CO2, ethanol is mixed with the SC-CO2. For example, several re- searchers used the supercritical reversed-phase evaporation method to prepare liposome aqueous dispersions by emulsification, by adding water to a homogeneous mixture of SC- CO2/phospholipid/ethanol with sufficient agitation, followed by decompression [18–21]. However, because ethanol is required when SC-CO2 is used as an alternative solvent, the problem of ethanol contamination of aqueous liposome solution arises again. In recent years, a method to prepare liposomes using SC-CO2 as a solvent, without using ethanol, by introducing ultrasonication has been investigated [22]. However, the disadvantage of SC-CO2 with ultrasonication is the risk of an accident when the bolts of the pressure vessel loosen owing to vibrations caused by ultrasonic irradiation. This is because the operating pressure is 10–20 MPa and leakage from inside the vessel must be avoided. In other words, the essence of the problem is the following. Organic solvents should be heated for evaporation; however, they have hazardous environmental impacts. SC-CO2 is nonpolar and its operating pressure is significantly high. Therefore, we thought that one solution would be to create liposomes using a slightly polar substance that has a significantly low boiling point, is a gas at room temperature, is easily volatile and is not toxic to living organisms. To satisfy these conditions, we focused on dimethyl ether (DME) as a candidate substance. Notably, DME is different from diethyl ether, which is commonly called ether, and its properties are also significantly different. DME is the simplest form of ether and has characteristics such as a low boiling point (–24.8 ◦C) [23]. Therefore, when DME is used as the extraction solvent, it is liquefied [24–30]. At room temperature, DME does not remain in the extracted materials [29,30]. The absence of the biological toxicity of DME has been confirmed by the bioassay evaluation of DME-dissolved water using a microbial culture [30]. DME is a safe extraction solvent for food production and has residue standards for food [31]. Furthermore, DME has excellent properties, such as zero ozone depletion potential and low global warming potential [32]. Therefore, DME has attracted attention as a new green solvent [33]. Additionally, DME has a bent molecular structure; therefore, it has weak polarity. For example, DME is known to form weak hydrogen bonds. DME dimers Foods 2021, 10, 1789 3 of 14

have small triply C−H···O−C improper bonds [34]. When water and liquefied DME come into contact, the oxygen of DME is the hydrogen-bond acceptor and the hydrogen of water is the hydrogen-bond donor. Therefore, liquefied DME can partially mix with water and its solubility in water is significantly low in its gaseous state [35,36]. Additionally, DME can extract polar substances from wet microalgae [37]. This indicates that phospholipids may also interact with liquefied DME. However, in recent years, attempts have been made to use this liquefied DME for purposes other than that of extraction medium; for example, it has been used as an antisolvent in amino acid crystallization [38], or a draw solute in forward osmosis [39]. To the best of our knowledge, no attempt has been made to apply liquefied DME to the preparation of liposomes. In this study, for the first time, we attempted to prepare liposomes by applying liquefied DME as a new medium; this is a method to avoid the problems of the high pressure of SC-CO2 and ethanol mixing in the liposome preparation. The expected operating pressure is approximately 0.5–0.6 MPa, which is much milder than that of SC-CO2 and there is no danger of the loosening of the pressure vessel because of ultrasound irradiation. The preparation method for liposomes is based on the simplest injection method. The properties of the DME-prepared liposomes were compared with those of the conventional method using diethyl ether with the same equipment. The properties and storage stability of the obtained liposomes were verified and the amount of residual media was verified.

2. Materials and Methods 2.1. Samples and Chemicals A mixture of soy lecithin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and cholesterol (90%; Wako Pure Chemical Industries, Ltd.) was used as the raw material for liposomes. Soy lecithin is a group of phospholipids mainly consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidic acid (PA) purified from soybeans. According to the manufacturer, the components were 38% of PC, 30% of PE, 25% of PA and 7% of other phospholipids. These are commonly used as raw materials for liposomes [40]. The phase transition temperature of the soybean lecithin was measured using a differential scanning calorimeter (DSC-60, Shimadzu Co., Kyoto, Japan) in a nitrogen atmosphere at a heating rate of 3 ◦C/min from 0 ◦C to 70 ◦C and it was 27.7 ◦C. Liquefied DME (supplied as a propellant filled in a spray-work air can 420D) was purchased from Tamiya, Inc., Shizuoka, Japan, and was used without further purification. Diethyl ether (Wako Pure Chemical Industries, Ltd.), the DME comparator, had a purity of 99.5%.

2.2. DME Injection Method A schematic diagram of the experimental setup for liposome preparation by the solvent injection method using liquefied DME as a medium is shown in Figure1. The apparatus consisted of a nitrogen cylinder, a stirring tank (96 mL, HPG96-3, Taiatsu Techno Corp., Saitama, Japan), 300 g of purified water in a glass beaker and an injection nozzle installed in the purified water. These were connected in series using 1/16-inch SUS tubes. The stirring tank consisted of a transparent, pressure-resistant glass container coated with polycarbonate. The stirring tank was filled with 60 mg of a mixture of soy lecithin and cholesterol in a 4:1 weight ratio and 40 ± 0.5 g of the liquefied DME or diethyl ether and it was stirred at 400 rpm and 23 ◦C for 20 min to ensure that the lipid mixture was completely dissolved in the medium. The internal pressure in the stirring tank was 0.56 MPa [23], which is the saturated vapor pressure for liquefied DME. An injection nozzle (2.5 mm in diameter) was placed 1.5 cm below the water surface in the glass beaker. The water temperature in the glass beaker was maintained at 60 ± 2 ◦C using a temperature controller and heater (300 W, CUNH-1103, Izumi Dennetsu Co., Ltd., Osaka, Japan). To clarify the effect of evaporation temperature, the slimmer test was conducted at 30 ◦C and 45 ◦C for comparison. Lipid-dissolved medium was injected into the water by pushing the surface of the medium in the stirred tank to the injection nozzle using nitrogen gas at 0.7 MPa. Foods 2021, 10, x FOR PEER REVIEW 4 of 14

Foods 2021, 10, 1789 effect of evaporation temperature, the slimmer test was conducted at 30 °C and 45 °C4 for of 14 comparison. Lipid-dissolved medium was injected into the water by pushing the surface of the medium in the stirred tank to the injection nozzle using nitrogen gas at 0.7 MPa. The injection speed was 10 mL/min and was adjusted using a manual flow rate valve by observingThe injection the decreasing speed was rate 10 mL/min of the medium and was level adjusted inside using the stirred a manual tank, flow using rate the valve vol- by observing the decreasing rate of the medium level inside the stirred tank, using the volume ume memory printed on the transparent wall of the stirred tank. The injected medium memory printed on the transparent wall of the stirred tank. The injected medium was was evaporated in water. The liposome dispersion was filtered using an aspirator (AS-01, evaporated in water. The liposome dispersion was filtered using an aspirator (AS-01, AS AS ONE Corp., Osaka, Japan) and filter paper (0.4 μm pore size, Kiriyama filter paper, ONE Corp., Osaka, Japan) and filter paper (0.4 µm pore size, Kiriyama filter paper, No. 5B, No. 5B, Kiriyama Glass Works Co., Tokyo, Japan) under reduced pressure to remove ex- Kiriyama Glass Works Co., Tokyo, Japan) under reduced pressure to remove excess lipids. cess lipids. Afterward, the liposome dispersion was decompressed in a rotary evaporator Afterward, the liposome dispersion was decompressed in a rotary evaporator (N-1200A, (N-1200A, EYELA, Tokyo, Japan) and a warm bath (SB-1200, EYELA) for 30 min to com- EYELA, Tokyo, Japan) and a warm bath (SB-1200, EYELA) for 30 min to completely pletely evaporate the medium. The obtained liposome dispersion was dialyzed for 3 days evaporate the medium. The obtained liposome dispersion was dialyzed for 3 days using a using a dialysis membrane (fractional molecular weight = 3500; Spectra/Por® 3, Repligen dialysis membrane (fractional molecular weight = 3500; Spectra/Por® 3, Repligen Japan, Japan, Ritto, Japan) to remove residual media from the aqueous phase outside the lipo- Ritto, Japan) to remove residual media from the aqueous phase outside the liposomes. The somes. The series of steps from liposome preparation to analysis was repeated three times series of steps from liposome preparation to analysis was repeated three times to check totheir check reproducibility. their reproducibility.

Figure 1. Schematic diagram of the liposome preparation system using liquefied DME. Figure 1. Schematic diagram of the liposome preparation system using liquefied DME. 2.3. Analysis 2.3.2.3.1. Analysis Transmission Electron Microscopy (TEM) Observation 2.3.1. TransmissionThe obtained liposomeElectron Microscopy dispersions were(TEM) dried Observation on a Cu microgrid for TEM observation (NP-C15,The obtained Okenshoji liposome Co., Ltd., dispersions Tokyo, Japan) were dried and observed on a Cu usingmicrogrid a TEM for (JEM2100H/FK,TEM observa- tionJEOL, (NP-C15, Ltd., Tokyo, Okenshoji Japan) at Co., an acceleration Ltd., Tokyo, voltage Japan) of 200 kV.Whenand observed the liposome using dispersiona TEM (JEM2100H/FK,was dried on the JEOL, TEM Ltd., microgrid, Tokyo, theJapan) liposomes at an acceleration were stained voltage using of the 200 negative kV. When staining the liposomemethod dispersion describedbelow was dried [41]. on In thethe negativeTEM microgrid, staining the method, liposomes the TEM were microgrids stained using were thehydrophilized negative staining by discharging method described 100 V ACbelow at 5[41]. A for In the 10 snegative using an staining electron method, microscope the hydrophilization system (DII-29020HD, JEOL, Ltd., Tokyo, Japan). A Parafilm® was floated TEM microgrids were◦ hydrophilized by discharging 100 V AC at 5 A for 10 s using an electronon the microscope water at 80 hydrophilizationC in a water bath system and (DII-29020HD, 0.5 mL of theliposome JEOL, Ltd., dispersion, Tokyo, Japan). purified A water and® 2 wt% phosphotungstic acid solution (Nacalai Tesque, Inc., Kyoto, Japan) were Parafilm was floated on the® water at 80 °C in a water bath and 0.5 mL of the liposome dispersion,dropped ontopurified the water Parafilm and .2 Thewt% hydrophilizedphosphotungstic TEM acid microgrid solution (Nacalai was contacted Tesque, with Inc., a droplet of liposome dispersion heated to 80 ◦C for 5 s. Subsequently, excess droplets Kyoto, Japan) were dropped onto the Parafilm®. The hydrophilized TEM microgrid was adhering to the TEM grid were removed by absorbing the water with a filter paper and contacted with a droplet of liposome dispersion heated to 80 °C for 5 s. Subsequently, the TEM grid was dried. Thereafter, a similar procedure was repeated with droplets of excess droplets adhering to the TEM grid were removed by absorbing the water with a purified water and 2 wt% phosphotungstic acid solution. Finally, the TEM microgrids were filter paper and the TEM grid was dried. Thereafter, a similar procedure was repeated dried in a desiccator for 12 h.

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Foods 2021, 10, 1789 with droplets of purified water and 2 wt% phosphotungstic acid solution. Finally,5 of the 14 TEM microgrids were dried in a desiccator for 12 h.

2.3.2. Dynamic Light Scattering (DLS) for Size Distribution and ζ-Potential Analysis 2.3.2. Dynamic Light Scattering (DLS) for Size Distribution and ζ-Potential Analysis The size distribution and ζ-potential of liposomes were measured at 25 °C using a ◦ DLS Theparticle size size distribution analyzer and (Zetasizerζ-potential Nano-Z of liposomesS, Malvern were Instruments measured Ltd., at 25 Worcestershire,C using a DLS particleUK). The size refractive analyzer indices (Zetasizer of liposomes Nano-ZS, and Malvern pure water Instruments were set Ltd., to 1.45 Worcestershire, and 1.33 for anal- UK). Theysis, refractive respectively indices [42,43]. of liposomes and pure water were set to 1.45 and 1.33 for analysis, respectively [42,43]. 2.3.3. Residual Medium Analysis 2.3.3. Residual Medium Analysis The liposome dispersion (1 mL) was placed in a gas chromatography (GC) headspace The liposome dispersion (1 mL) was placed in a gas chromatography (GC) headspace vial and 1 mL of a surfactant, Tween®20 (polyoxyethylene (20) sorbitan monolaurate) so- vial and 1 mL of a surfactant, Tween®20 (polyoxyethylene (20) sorbitan monolaurate) solutionlution (10 (10 wt%, wt%, Tokyo Tokyo Chemical Chemical Industry Industry Co Co.,., Ltd., Ltd., Tokyo, Tokyo, Japan) Japan) was was added. Afterward, thethe mixturemixture waswas vigorouslyvigorously shakenshaken and allowedallowed to stand for 10 min to disrupt the lipo- somessomes [[44].44]. Thus, thethe residualresidual mediummedium insideinside thethe liposomeliposome waswas released.released. TheThe releasedreleased mediummedium was evaporatedevaporated by holdingholding atat 5555 ◦°CC forfor 3030 minmin inin aa vial.vial. UsingUsing aa microsyringemicrosyringe heatedheated toto 6060 ◦°C,C, 100 µμLL ofof thethe gasgas phasephase waswas obtainedobtained fromfrom thethe vialvial andand analyzedanalyzed byby GC.GC. FollowingFollowing aa previousprevious studystudy [[45,46],45,46], thethe amount ofof residual mediamedia waswas analyzedanalyzed usingusing GCGC coupledcoupled withwith aa ﬂameflame ionizationionization detectordetector (GC-2014,(GC-2014, ShimadzuShimadzu Co.,Co., Kyoto,Kyoto, Japan).Japan). AA methylmethyl polysiloxanepolysiloxane capillarycapillary column column (HP-5MS; (HP-5MS; 30 30 m m× ×250 250µ μmm (internal(internal diameter) diameter)× × 0.250.25 µμm,m, AgilentAgilent TechnologiesTechnologies Tokyo Tokyo Ltd., Ltd.) Tokyo, was em Japan)ployed. was The employed. oven temperature The oven temperaturewas initially wasset at initially 100 °C setfor at45 100min◦ Cand for increased 45 min and to 160 increased °C at a to rate 160 of◦ C10 at °C/min. a rate ofThe 10 inlet◦C/min. tempera- The inletture was temperature 200 °C and was the 200 detection◦C and thetemperature detection was temperature 160 °C. was 160 ◦C. TheThe amountamount ofof residualresidual mediamedia waswas alsoalso measuredmeasured whenwhen thethe dialysisdialysis membranemembrane waswas notnot used,used, becausebecause the the removal removal of of excess excess media media from from the the liposome liposome by the by dialysisthe dialysis membrane mem- wasbrane time-consuming. was time-consuming. In this case,In this the case, media the wasmedia removed was removed only by only decompression by decompression using a rotaryusing a evaporator rotary evaporator and the decompression and the decompression time was changedtime was to changed 0, 10 and to 20 0, min 10 and to measure 20 min theto measure change overthe change time. Theover pretreatment time. The pretreatment conditions conditions using Tween20 using® Tween20and the detection® and the conditionsdetection conditions using GC wereusing theGC same. were the same.

3.3. Results TheThe liposome dispersionsdispersions preparedprepared usingusing liqueﬁedliquefied DMEDME andand diethyldiethyl etherether areare shownshown inin FigureFigure2 2.. The The prepared prepared liposome liposome solution solution had had a a white white transparent transparent dispersion dispersion in in both both methods.methods. The following analyses werewere performedperformed onon thesethese aqueousaqueous liposomeliposome solutions.solutions.

FigureFigure 2.2. Liposome dispersions prepared usingusing liqueﬁedliquefied DMEDME ((leftleft)) andand diethyldiethyl etherether ((rightright).).

3.1. TEM Observation Figure3 shows the TEM images of the liposomes prepared using liqueﬁed DME at ◦ 60 C, which is the typical image observed by negative staining [41]. The boundary between Foods 2021, 10, x FOR PEER REVIEW 6 of 14

Foods 2021, 10, 1789 6 of 14 3.1. TEM Observation Figure 3 shows the TEM images of the liposomes prepared using liquefied DME at 60 °C, which is the typical image observed by negative staining [41]. The boundary be- thetween liposome the liposome and its surroundings,and its surroundings, the structure the structure of the lipid of the membrane lipid membrane and the portion and the of theportion liposome of the interior liposome caused interior by thecaused aqueous by the phase aqueous were phase observed. were observed. The black The contrast black in thecontrast central in partthe central was due part to was the scatteringdue to the of scattering electron beamsof electron by the beams phosphotungstic by the phospho- acid thattungstic penetrated acid that the penetrated liposome the during liposome staining, during indicating staining, that indicating the aqueous that phasethe aqueous existed beforephase existed drying before [41]. As drying shown [41]. in As Figure shown3a, in we Figure observed 3a, we liposomes observed with liposomes a diameter with ofa approximatelydiameter of approximately 60 nm and 60 a tracenm and of a an trace aqueous of an aqueous phase in phase the center. in the center. Figure Figure3b shows 3b liposomesshows liposomes with a with diameter a diameter of approximately of approximately 150 nm 150 and nm aand dense a dense lipid lipid membrane, membrane, with approximatelywith approximately 10 layers 10 layers with awith thickness a thickness of 4 nm, of 4 as nm, shown as shown in Figure in Figure3d. Figure 3d. Figure3c shows 3c liposomesshows liposomes with a with diameter a diameter of approximately of approximately 170 nm, 170 a nm, trace a oftrace an aqueousof an aqueous phase phase in the center.in the center. Since the Since typical the typical thickness thickness of lipid of membrane lipid membrane is 4–6 nm, is 4–6 this nm, layer this can layer be regarded can be asregarded the lipid as membrane the lipid membrane of liposomes. of liposomes.

FigureFigure 3.3. TEM images of of the the liposomes liposomes prepared prepared by by the the liquefied liqueﬁed DME DME method method at 60 at °C. 60 Magnification◦C. Magniﬁcation (a) × 100 (a) ×k, (100b) × k, (b200) × k200 and k ( andc) × 300 (c) × k. 300(d) Enlarged k. (d) Enlarged view of view (b), ofwith (b), emphasis with emphasis on clarity on clarityand contrast. and contrast.

Figure4 4 showsshows the TEM images images of of the the lipos liposomesomes prepared prepared using using diethyl diethyl ether ether at 60 at 60°C.◦ InC. Figure In Figure 4a, 4aa, LUV a LUV with with a single a single lipid lipidmembrane membrane was observed. was observed. In Figure In Figure4b, MLVs4b, MLVswith traces with tracesof aqueous of aqueous phase phasein the incenter the centerwere observed. were observed. In Figure In Figure4c, liposomes4c, liposomes with withlipid lipidmembranes membranes ranging ranging from from50 to 125 50 to nm 125 were nm observed were observed and the and thickness the thickness of the lipid of the lipidmembrane membrane was approximately was approximately 6 nm. 6 The nm. TEM The TEMimages images of these of theseliposomes liposomes are not are so not dif- so differentferent from from those those of the of the liposomes liposomes prepared prepared using using liquefied liqueﬁed DME DME (Figure (Figure 3). 3).

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Figure 4. TEM images of the liposomes prepared by the conventional diethyl ether method at 60 °C. Magnification◦ (a) × FigureFigure 4. 4. TEM images images of of the the liposomes liposomes prepared prepared byby the the conventional conventional diethyl diethyl ether ether method method at 60 at°C. 60 MagnificationC. Magnification (a) × 100 k, (b) × 200 k and (c) × 300 k. (a100) × k,100 (b k,) × (200b) × k 200andk (c and) × 300 (c) ×k. 300 k. Figure 5 shows the TEM images of the liposomes prepared using liquefied DME and FigureFigure5 5 shows shows thethe TEMTEM imagesimages ofof the liposomes prepared using liquefied liquefied DME DME and and diethyl ether at 45 ◦°C. For the liquefied DME (Figure 5a,b), liposomes of approximately diethyldiethyl etherether at 45 °C.C. For the liquefiedliquefied DM DMEE (Figure 55a,b),a,b), liposomes of approximately 100 nm and 2–3 lipid layers with a thickness of approximately 5 nm were observed. For 100100 nmnm andand 2–32–3 lipidlipid layerslayers with a thicknessthickness of approximately 5 5 nm nm were were observed. observed. For For diethyl ether (Figure 5c,d), liposomes with lipid films in the range of 50–100 nm were diethyldiethyl etherether (Figure 55c,d),c,d), liposomesliposomes withwith lipidlipid filmsfilms in the range of 50–100 nm were observed. Liposomes with traces of the aqueous phase were observed for the liquefied observed.observed. Liposomes with with traces traces of of the the aq aqueousueous phase phase were were observed observed for for the the liquefied liquefied DME and diethyl ether. The TEM images of these liposomes were not very different from DMEDME andand diethyl ether. The TEM images of of th theseese liposomes liposomes were were not not very very different different from from those of the liposomes prepared at 60 ◦°C (Figures 3 and 4). thosethose ofof thethe liposomesliposomes prepared at 60 °CC (Figures (Figures 3 andand4 4).).

Figure 5. TEM images of the liposomes prepared by (a,b) the liquefied DME and (c,d) diethyl ether method at 45 °C. FigureFigure 5. 5. TEMTEM imagesimages ofof thethe liposomesliposomes prepared by ( a,,b)) the the liquefied liquefied DME DME and and ( (cc,d,d)) diethyl diethyl ether ether method method at at 45 45 °C.◦C. Magnification × 200 k. MagnificationMagnification× × 200 k. FigureFigure6 6 shows shows thethe TEMTEM imagesimages ofof thethe liposomesliposomes preparedprepared using liquefied liquefied DME at at Figure 6 shows the TEM images of the liposomes prepared using liquefied DME at 3030 ◦°C.C. Notably, the the boiling boiling point point of of diethyl diethyl ether ether is 34.8 is 34.8 °C; ◦therefore,C; therefore, liposomes liposomes could could not 30 °C. Notably, the boiling point of diethyl ether is 34.8 °C; therefore, liposomes could not notbe created be created at 30 at °C, 30 ◦thusC, thus TEM TEM observation observation was wasnot performed. not performed. When When prepared prepared with withliq- be created at 30 °C, thus TEM observation was not performed. When prepared with liq- liquefieduefied DME DME at at30 30°C,◦ C,lipid lipid aggregates aggregates (Fig (Figureure 6a)6 anda) and liposomes liposomes (Figure (Figure 6b)6 wereb) were ob- uefied DME at 30 °C, lipid aggregates (Figure 6a) and liposomes (Figure 6b) were ob- observed.served. The The aggregates aggregates shown shown in inFigure Figure 6a6 ahad had a a diameter diameter of of 600–700 600–700 nm nm and and rod-like rod-like served. The aggregates shown in Figure 6a had a diameter of 600–700 nm and rod-like structuresstructures withwith aa length length of of 30–100 30–100 nm nm were were observed observed inside inside the the aggregates. aggregates. In In Figure Figure6b, 6b, in structures with a length of 30–100 nm were observed inside the aggregates. In Figure 6b, additionin addition to thoseto those with with a diameter a diameter of of approximately approximately 100 100 nm, nm, similar similar to to those those observed observed in in addition to those with a diameter of approximately 100 nm, similar to those observed thein the preparations preparations at 60at 60◦C °C and and 45 45◦C, °C, several several small small spherical spherical materials materials with with a diametera diameter of in the preparations at 60 °C and 45 °C, several small spherical materials with a diameter approximatelyof approximately 30–50 30–50 nm, nm, which which may may not not be be liposomes, liposomes, were were observed. observed. of approximately 30–50 nm, which may not be liposomes, were observed.

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Figure 6. TEM images of the liposomes prepared by the liquefied DME method at 30 °C. Magnifica- tion (a) × 100 k and (b) × 200 k.

3.2. DLS for Size Distribution and ζ-Potential Analysis Figure 7 shows the DLS size distributions of the liposomes obtained using liquefied DMEFigureFigure and 6.6. TEMdiethyl images ether. of There the liposomes was no preparedsignificant byby differencethethe liquefiedliquefied in DMEDME the size methodmethod distributions atat 3030 ◦°C.C. Magnifica-Magnifica- of the liquefiedtiontion ((aa)) ×× DME100100 k k andand ( (diethylbb)) ×× 200200 etherk. k. liposomes and the distribution of the liposomes ranged from 60 to 350 nm. At 60 °C, the average liposome sizes were 153 nm for liquefied DME and3.2.3.2. 162 DLS nm forfor for SizeSize dimethyl DistributionDistribution ether, andand indicating ζζ-Potential-Potential no AnalysisAnalysis significant difference. The polydispersity index Figure(PDI)Figure of7 7 showsthese shows liposomes the the DLS DLS size wassize distributions 0.12distributions ± 0.01 for of bothof the the liquefied liposomes liposomes DME obtained obtained and dimethyl using using liquefied liquefiedether. TheseDMEDME values andand diethyldiethyl are smaller ether.ether. than ThereThere the waswas PDI nono after significantsignificant conversion differencedifference of heterogeneous inin thethe sizesize distributionsliposomesdistributions to ho- ofof thethe mogeneous liposomes in the microchannel laminar flow with sonication [47], indicating liquefiedliquefied DMEDME andand diethyldiethyl etherether liposomesliposomes andand thethe distributiondistribution ofof thethe liposomesliposomes rangedranged that very homogeneous liposomes◦ were prepared by injection method using liquefied fromfrom 6060 toto 350350 nm.nm. AtAt 6060 °C,C, thethe averageaverage liposomeliposome sizessizes werewere 153153 nmnm forfor liquefiedliquefied DMEDME DME. The diameters of the liposomes obtained by DLS were generally consistent with andand 162162 nmnm forfor dimethyldimethyl ether,ether, indicatingindicating nono significantsignificant difference.difference. TheThe polydispersitypolydispersity those from the TEM observations, confirming the validity of the results. indexindex (PDI)(PDI) ofof thesethese liposomesliposomes waswas 0.120.12 ±± 0.010.01 for for both both liquefied liquefied DME DME and and dimethyl dimethyl ether. Conversely, when the liposomes were prepared using liquefied DME at 30 °C, the TheseThese values are smaller smaller than than the the PDI PDI after after conversion conversion of ofheterogeneous heterogeneous liposomes liposomes to ho- to size distribution by DLS showed a medium liposome size of 78 nm and a large number of homogeneousmogeneous liposomes liposomes in in the the microchannel microchannel lami laminarnar flow flow with with sonicati sonicationon [[47],47], indicatingindicating smaller materials were produced. It was also implied that there was a small amount of thatthat veryvery homogeneous homogeneous liposomes liposomes were were prepared prepared by injectionby injection method method using using liquefied liquefied DME. material in the 100–300 nm range. These results were also consistent with a large number TheDME. diameters The diameters of the liposomes of the liposomes obtained obtain by DLSed were by DLS generally were consistentgenerally withconsistent those fromwith of small clumps and large aggregates observed in the TEM images. thethose TEM from observations, the TEM observations, confirming theconfirming validity the of the validity results. of the results. Conversely, when the liposomes were prepared using liquefied DME at 30 °C, the size distribution by DLS showed a medium liposome size of 78 nm and a large number of smaller materials were produced. It was also implied that there was a small amount of material in the 100–300 nm range. These results were also consistent with a large number of small clumps and large aggregates observed in the TEM images.

Figure 7. Diameter distribution of the liposome prepared using liqueﬁed DME and diethyl ether. Figure 7. Diameter distribution of the liposome prepared using liquefied DME and diethyl ether. Conversely, when the liposomes were prepared using liqueﬁed DME at 30 ◦C, the size distribution by DLS showed a medium liposome size of 78 nm and a large number of smaller materials were produced. It was also implied that there was a small amount of material in the 100–300 nm range. These results were also consistent with a large number

of small clumps and large aggregates observed in the TEM images. FigureThe 7. Diameterζ-potentials distribution of the liposomeof the liposome prepared prepared using using liqueﬁed liquefied DME DME andand diethyl ether. ether were −57.2 and −57.9 mV and no signiﬁcant difference was observed. In general, particles

Foods 2021, 10, x FOR PEER REVIEW 9 of 14

Foods 2021, 10, 1789 9 of 14 The ζ-potentials of the liposome prepared using liquefied DME and diethyl ether were −57.2 and −57.9 mV and no significant difference was observed. In general, particles are stable above 30 mV or below −30 mV in their dispersion [48]. Therefore, these DME liposomesare stable had above a negative 30 mV orpotential below −and30 mVwere in dispersed their dispersion with high [48]. repulsion Therefore, among these the DME liposomes.liposomes In hadother a negativewords, the potential size and and dispersive were dispersed stability of with the high liposomes repulsion obtained among us- the ingliposomes. liquefied DME In other were words, comparable the size to those and dispersive obtained using stability the ofconventional the liposomes technique, obtained suggestingusing liquefied that liposomes DME were can comparable be successfully to those prepared. obtained using the conventional technique, suggesting that liposomes can be successfully prepared. The ζ-potentials of the liposomes prepared using liquefied DME and diethyl ether at The ζ-potentials of the liposomes prepared using liquefied DME and diethyl ether at 45 °C were −62.4 and −60.5 mV and that of the liposomes prepared using liquefied DME 45 ◦C were −62.4 and −60.5 mV and that of the liposomes prepared using liquefied DME at 30 °C was −58.8 mV. at 30 ◦C was −58.8 mV.

3.3.3.3. Residual Residual Medium Medium Analysis Analysis AfterAfter the the final final treatment treatment with with the dialysis the dialysis membrane, membrane, the residual the residual medium medium could couldnot be notdetected be detected by GC byin GCthe inliposomes the liposomes prepared prepared using usingliquefied liquefied DME. DME.This means This means that the that residualthe residual solvent solvent was less was than less 0.2 than mg/L. 0.2 mg/L.Conversely, Conversely, in the inliposomes the liposomes prepared prepared using usingdi- ethyldiethyl ether, ether, there there was was0.683 0.683 mg/L mg/L of residual of residual solvent solvent in the in liposomes. the liposomes. WithoutWithout the the final final treatment treatment using using the the dial dialysisysis membrane, membrane, before before the the decompression decompression treatmenttreatment using using the the rotary rotary evaporator evaporator immedi immediatelyately after after liposome liposome preparation, preparation, the the resid- resid- ualual media media in inthe the entire entire aqueous aqueous liposome liposome solution solution prepared prepared usin usingg liquefied liquefied DME DME was was 18201820 mg/L mg/L and and that that using using diethyl diethyl ether ether was was 610 61000 mg/L, mg/L, as shown as shown in Figure in Figure 8. 8After. After 10 10 min min of ofdecompression, decompression, the the residual residual DME DME rapidly rapidly decreased decreased to to 40.7 40.7 mg/L. mg/L. Similarly, Similarly, the the resid- residual ualdiethyl diethyl ether ether concentrationconcentration waswas 383 mg/L. After After 20 20 min min of of decompression, decompression, the the residual residual DMEDME decreased decreased to toa relatively a relatively low low limit limit of ofdetection detection (<0.2 (<0.2 mg/L). mg/L). In Incontrast, contrast, the the residual residual diethyldiethyl ether ether was was detected detected to tobe be95.8 95.8 mg/L. mg/L.

Figure 8. Residual media in aqueous liposome solution for only decompression treatment without Figure 8. Residual media in aqueous liposome solution for only decompression treatment without dialysis membrane. dialysis membrane. 3.4. Stability in a Large Storage Period 3.4. Stability in a Large Storage Period In order to verify the long-term storage stability of liposomes prepared with liquefied DME,In order they to were verify stored the long-term at 4 ◦C and storage 37 ◦C forstability up to of 21 liposomes days and theprepared medium with diameters liquefied and DME,ζ-potentials they were were stored measured at 4 °C and by DLS37 °C at fo variousr up to 21 storage days and times. the The medium results diameters on the stability and ζ-potentialsof liposomes were stored measured at 4 ◦ byC andDLS 37 at ◦variousC are shown storage in times. Figures The9 and results 10, on respectively. the stability As of shownliposomes in Figuresstored 9at and 4 °C 10 and, the 37 average °C are diametershown in and Figuresζ-potential 9 and 10, of liposomesrespectively. did As not shownchange in significantlyFigures 9 and when 10, storedthe average at 4 ◦C diameter or 37 ◦C, indicatingand ζ-potential that liposomes of liposomes prepared did not using liquefied DME as a medium were stably dispersed for a long time.

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Foods 2021, 10, 1789 10 of 14 changechange significantlysignificantly whenwhen storedstored atat 44 °C°C oror 3737 °C,°C, indicatingindicating thatthat liposomesliposomes preparedprepared us-us- inging liquefiedliquefied DMEDME asas aa mediummedium werewere stablystably disperseddispersed forfor aa longlong time.time.

Figure 9. Medium diameter and ζ-potential of liposomes prepared with liqueﬁed DME and stored FigureFigure 9.9. MediumMedium diameterdiameter andand ζζ-potential-potential ofof liposomesliposomes preparedprepared withwith liquefiedliquefied DMEDME andand storedstored at 4 ◦C. atat 44 °C.°C.

Figure 10. Medium diameter and ζ-potential of liposomes prepared with liquefied DME and stored FigureFigure 10.10. MediumMedium diameterdiameter andand ζζ-potential-potential ofof liposomesliposomes preparedprepared withwith liquefiedliquefied DMEDME andand storedstored at 37 ◦C. atat 3737 °C.°C. 4. Discussion 4.4. DiscussionDiscussion In this study, liposomes were prepared using liquefied DME instead of conventional organicInIn thisthis solvents, study,study, liposomesliposomes which are werewere toxic preparedprepared and difficult usingusing to liquefiedliquefied handle andDMEDME they insteadinstead cause ofof aconventionalconventional large environ- organicorganic solvents,solvents, whichwhich areare toxictoxic andand difficuldifficultt toto handlehandle andand theythey causecause aa largelarge environ-environmental impact and religious problems [7–9], and SC-CO2, which requires the addition of mentalmentalethanol impactimpact [18–21 andand], high religiousreligious operating problemsproblems pressure [7–9],[7–9], and andand sonication SC-COSC-CO22,, whichwhich to dissolve requiresrequires phospholipids thethe additionaddition ofof [22 ]. ethanolethanolFirst, the [18–21],[18–21], TEM observationhighhigh operatingoperating and pressurepressure DLS measurements andand sonicationsonication of size toto dissolvedissolve and ζ-potential phospholipidsphospholipids showed [22].[22]. that it First,First,was possiblethethe TEMTEM to observationobservation prepare liposomes andand DLSDLS with measurementsmeasurements almost the sameofof sizesize properties andand ζζ-potential-potential as those showedshowed of conventional thatthat itit waswasdiethyl possiblepossible ether, toto even prepareprepare when liposomesliposomes using liquefied withwith almostalmost DME. thethe The samesame pressure propertiesproperties in the asas vessel thosethose of ofof the conven-conven- liquefied tionaltional diethyldiethyl ether,ether, eveneven whenwhen usingusing liquefliquefiedied DME.DME. TheThe pressurepressure inin thethe vesselvessel ofof thethe DME is 0.56 MPa, which is considerably lower than the operating pressure of SC-CO2 liquefiedliquefied(10–20 MPa) DMEDME [ 13 isis – 0.560.5622]. MPa,MPa, The high whichwhich safety isis considerablyconsiderably of DME can lowerlower be seen thanthan from thethe the operatingoperating fact that pressurepressure the container ofof SC-COSC-COused in22 (10–20(10–20 this test MPa)MPa) was [13–22].[13–22]. made of TheThe glass. highhigh safetysafety ofof DMEDME cancan bebe seenseen fromfrom thethe factfact thatthat thethe containercontainerThe usedused temperature inin thisthis tete ofstst thewaswas pure mademade water ofof glass.glass. into which the medium is injected should be 45 ◦C or higher. It cannot be denied that liposomes can be created at water temperatures between 30 and 45 ◦C. Conversely, at 30 ◦C, which is well above the boiling point of liquefied DME, the aggregates of lipids other than liposomes were observed by TEM because of the phase Foods 2021, 10, 1789 11 of 14

transition temperature of phospholipids. Phospholipid bilayers become more flexible, fluid and permeable at temperatures above the phase transition temperature. Therefore, it is preferable to prepare liposomes at or above the phase-transition temperature [49]. The phase transition temperature of the soybean lecithin used in this experiment was 27.7 ◦C, as mentioned in Section 2.1, which is close to the injection temperature of 30 ± 2 ◦C. Although the pure water was accurately controlled, the heat of vaporization may cause a local decrease in temperature near the injection nozzle, where the liquefied DME vaporizes. This local temperature decrease may have inhibited liposome formation because the lipid membrane was not sufficiently flexible. The amount of residual medium was measured by GC and, for diethyl ether, the residual medium was observed even after the medium was removed using decompression and dialysis membrane treatment. This suggests that the medium remained inside the liposome, which is a problem in terms of biotoxicity and transporting functional materials into the body. In contrast, it was suggested that liquefied DME can completely remove the residual medium from the inside of the liposome without using a dialysis membrane, just by a 20-minute decompression operation. In addition to the fact that DME-dissolved water has considerably low biotoxicity [30], the result showing that the residual amount was below the detection limit indicates that the method of preparing liposomes using liquefied DME is superior to that using conventional organic solvents in terms of biocompatibility. Additionally, previous studies have shown that liquefied DME can extract a variety of functional substances, such as astaxanthin [50,51], fucoxanthin [52], lutein [29,53], resveratrol and its glycosides [54], catechins and caffeine [55], tocopherol [56], gingerols and capsaicin [27]. Among these substances, carotenoids, such as astaxanthin, fucoxanthin and lutein, are hydrophobic, therefore difficult to encapsulate into liposomes by mechanical methods. Therefore, a medium to dissolve them is necessary to encapsulate them into liposomes. Moreover, the solubility of these carotenoids in SC-CO2 is so extremely low that SC-CO2 is used as an anti-solvent for crystallizing carotenoids [57–59]. In the future, these functional substances and natural pigments that are difficult to handle with SC-CO2 are expected to be dissolved in liquid DME together with soy lecithin and cholesterol and injected into warm water to be encapsulated in liposomes.

5. Conclusions In this study, liposomes were prepared by dissolving soy lecithin and cholesterol in liquefied DME and injecting them into warm water. As a result of the TEM image observation, size distribution measurement by DLS and ζ-potential measurement, it was found that the liquefied DME injection method succeeded in producing liposomes similar to those produced using conventional diethyl ether at above 45 ◦C. We also measured the amount of residual DME and found that DME was no longer detected after only 20 min of the depressurization of the liposome dispersion. In contrast, with conventional diethyl ether, a large amount of diethyl ether remained even after depressurization and dialysis membrane treatment. Considering the considerably low boiling point of DME and the low toxicity of DME to living organisms, even if it remains, the safety of the liquefied DME injection method is much higher than that of the conventional method. Furthermore, the operating pressure of the liquefied DME injection method is much lower than that of SC- CO2, which is also known as a green medium, and ethanol is not required. In other words, in comparison with SC-CO2, DME is a superior medium in terms of ethanol contamination and safety during operation.

Author Contributions: Conceptualization, H.K.; data curation, T.K.; funding acquisition, H.K.; investigation, H.K., T.K. and W.; methodology, H.K., T.K. and W.; project administration, H.K.; resources, H.K.; supervision, M.G.; validation, H.K.; visualization, H.K. and T.K.; writing—original draft, H.K. and T.K.; writing—review and editing, H.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI (grant number JP20K05192 to H.K.). Foods 2021, 10, 1789 12 of 14

Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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