life

Article Giant Vesicles Produced with (PCs) and (PEs) by Water-in-Oil Inverted Emulsions

Boying Xu 1,† , Jinquan Ding 2,†, Jian Xu 1,* and Tetsuya Yomo 1,*

1 Laboratory of Biology and Information Science, Biomedical Synthetic Biology Research Center, School of Life Sciences, East China Normal University, Shanghai 200062, China; [email protected] 2 School of Software Engineering, East China Normal University, Shanghai 200062, China; [email protected] * Correspondence: [email protected] (J.X.); [email protected] (T.Y.); Tel.: +86-21-62233727 (J.X. & T.Y.) † These authors contributed equally to this work.

Abstract: (1) Background: giant vesicles (GVs) are widely employed as models for studying physic- ochemical properties of bio-membranes and artificial cell construction due to their similarities to natural cell membranes. Considering the critical roles of GVs, various methods have been developed to prepare them. Notably, the water-in-oil (w/o) inverted emulsion-transfer method is reported to be the most promising, owning to the relatively higher productivity and better encapsulation efficiency of biomolecules. Previously, we successfully established an improved approach to acquire detailed information of 1-Palmitoyl-2-oleoyl-sn-glycero-3- (POPC)-derived GVs with imaging

 flow cytometry (IFC); (2) Methods: we prepared GVs with different compositions, including  phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and PC/PE mixtures by w/o in-

Citation: Xu, B.; Ding, J.; Xu, J.; verted emulsion methods. We comprehensively compared the yield, purity, size, and encapsulation Yomo, T. Giant Vesicles Produced efficiency of the resulting vesicles; (3) Results: the relatively higher productivities of GVs could be with Phosphatidylcholines (PCs) and obtained from POPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3- Phosphatidylethanolamines (PEs) by phosphoethanolamine (DLPE), DOPC: DLPE (7:3), and POPC: DLPE (6:4) pools. Furthermore, we Water-in-Oil Inverted Emulsions. Life also demonstrate that these GVs are stable during long term preservation in 4 ◦C. (4) Conclusions: 2021, 11, 223. https://doi.org/ our results will be useful for the analytical study of GVs and GV-based applications. 10.3390/life11030223 Keywords: giant vesicles; water-in-oil inverted emulsions; IFC; lipid; membrane Academic Editor: Pasquale Stano

Received: 9 February 2021 Accepted: 2 March 2021 1. Introduction Published: 10 March 2021 To understand the organization and dynamics of lipid bilayers, research on giant

Publisher’s Note: MDPI stays neutral vesicles (GVs) as an artificial cellular membrane model has gained considerable interest in with regard to jurisdictional claims in recent decades [1–6]. GVs consisting of membranes are similar to modern published maps and institutional affil- cellular membranes in terms of physical and chemical properties. Additionally, GVs have iations. large dimensions of their typical cell sizes, making them suitable artificial compartments for the construction of synthetic cells [7,8] through the bottom-up approach [9], is very attractive in the synthetic biology field. Thus, the development of reliable protocols for the preparation and analysis of GVs in a designed environment is crucial for promoting the synthetic cell project to the next stage. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. The importance of GVs has initiated a massive effort in developing an easy and ef- This article is an open access article ficient preparation method to produce vesicles from known lipid compositions [6]. The distributed under the terms and challenge lies in establishing a reproducible way to produce GVs with a considerable encap- conditions of the Creative Commons sulation efficacy for biomolecules in a desired yield and quality. To date, various techniques Attribution (CC BY) license (https:// for GV preparations have been established, such as lipid hydration including simple film creativecommons.org/licenses/by/ hydration [10] and gel- [11] or paper-assisted hydration [12], electro-formation [13,14], and 4.0/). emulsion phase transfer method [15–17]. Lipid hydration, as one of the most commonly

Life 2021, 11, 223. https://doi.org/10.3390/life11030223 https://www.mdpi.com/journal/life Life 2021, 11, 223 2 of 13

used approaches, is easy to handle and only requires several common facilities and equip- ment, such as an evaporator and vacuum pump. However, it always requires experimental optimizations, and the resulting vesicles usually have polydisperse size distributions and low encapsulation efficiencies. Comparatively, GVs from the electro-formation method could produce uniform and uni-lamellar GVs, possessing a high reproducibility and lower polydispersity. Unfortunately, low yield, low encapsulation efficiency and limited lipid compositions are the main drawbacks of the electro-formation method. The microfluidic technique has recently been demonstrated as a state-of-art method of preparing GVs [18–21] in a designed micro-chip. Although a microfluidic chip does yield monodisperse GVs with higher encapsulation efficiencies in precise control over production, it often requires unique equipment setups and expertise in designing an appropriate chip, which is not always available to researchers [22–24]. Compared to the methods mentioned above, phase transfer methods such as the water-in-oil (w/o) emulsion transfer, has shown significant advantages, since it does not need specialized equipment, is of straightforward implemen- tation, acceptable encapsulation efficiency, and high yield of uni-lamellar vesicles [15,16], although one drawback is that there is a possible presence of oil trapped within the bilayers depending on the protocols and reagent used, which might influence membrane mechan- ical properties [17,25,26]. Besides, this method can also produce GVs with a wide range of lipid compositions, e.g., phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), which are usually employed for constructing the artificial [6,27,28]. As we know, PCs and PEs are major components in cell membranes and they are preferentially located in different leaflets of the bilayer: the outer layer for PCs and the inner for PEs [29]. Some studies have also suggested that the PC/PE ratio is a critical modulator of membrane integrity [30]. To date, many studies have already reported the productions of vesicles made from PC/PE [6]. For example, PC or PE mixed with 10–20 mol% of a charged lipid (e.g., , CL; , PA; , PG; , PS) can form GVs through the gentle hydration method [31]; By the electro-formation method, GVs can be prepared with non-charged lipids [32] like PCs (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC [33]; 1,2-dimyristoyl-sn-glycero-3- phosphocholine, DMPC [34] or egg yolk phosphatidylcholines, egg PC [35]); DOPC or egg PC can produce GVs by the w/o emulsion transfer method [36,37]. Morphological analysis of vesicles using electron microscopy techniques (SEM or TEM) is quite time- consuming and needs expensive machines and regular maintenances. Moreover, traditional microscopy observation sometimes does not precisely reflect the real results, e.g., size distribution and aspect ratio of particles. Finally, dynamic light scattering (DLS) can readily provide particle size and polydispersity index, but it is most suited for small and large liposomes, not micrometric ones [38]. DLS analysis does not provide visualized information of particles and usually mistakenly treats aggregated vesicles as one single particle. It is known that the flow cytometer (FCM) technology is convenient for analyzing GVs with statistically relevant measurements in large populations. More recently, imaging FCM (IFC) has been developed to analyze a large number of features based on morphology like size, shape, fluorescent intensity, circularity, and texture on a specific cell or compartment of a cell [39,40]. Our previous study established a straightforward protocol to investigate 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-derived GVs quantitatively and qualitatively on Amnis ImageStream Mark II [15]. Continuing that effort in this study, we report the preparation of GVs with different lipid compositions such as PC, PE, and PC-PE mixtures in different ratios by the water-in- oil inverted emulsion method. Besides, we analyze and compare the yield, purity, size, and encapsulation efficiency of resulting vesicles via IFC analysis. Our work shows that PCs (POPC, DOPC, DMPC), PEs (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine, DLPE; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), and PC:PE mixtures (POPC: DLPE, DOPC: DLPE, and DMPC: DLPE) can be utilized to yield high production of GVs with significantly high encapsulation efficiency. Importantly, we also find that most of these GVs from w/o emulsion method show high stability over long incubation times at Life 2021, 11, x FOR PEER REVIEW 3 of 16

Life 2021, 11, x FOR PEER REVIEW 3 of 16

Life 2021, 11, x FOR PEER REVIEW 3 of 16

Life 2021, 11, x FOR PEER REVIEW PCs (POPC, DOPC, DMPC), PEs (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,3 of 16

Life 2021, 11, x FOR PEER REVIEW DLPE;PCs (POPC, 1,2-dimyristoyl-sn-glycero-3-phospho DOPC, DMPC), PEs (1,2-dilaurethanolamine,oyl-sn-glycero-3-phosphoethanolamine, DMPE), and PC:PE mixtures3 of 16

Life 2021, 11, x FOR PEER REVIEW (POPC:PCsDLPE; (POPC, 1,2-dimyristoyl-sn-glycero-3-phospho DLPE, DOPC,DOPC: DLPE,DMPC), and PEs DMPC: (1,2-dilaur DLPE)ethanolamine, oyl-sn-glycero-3-phosphoethanolamine,can be utilized DMPE), to yield and highPC:PE production mixtures3 of 16

ofDLPE;(POPC:PCs GVs (POPC, 1,2-dimyristoyl-sn-glycero-3-phospho withDLPE, significantly DOPC,DOPC: DLPE,DMPC), high and encapsulationPEs DMPC: (1,2-dilaur DLPE)ethanolamine, efficiency. oyl-sn-glycero-3-phosphoethanolamine,can be utilized Importantly, DMPE), to yield and we highPC:PE also production findmixtures that PCsmost(POPC:ofDLPE; GVs (POPC,of 1,2-dimyristoyl-sn-glycero-3-phospho withDLPE,these significantlyGVs DOPC,DOPC: from DLPE,DMPC), w/o high emulsionand encapsulationPEs DMPC: (1,2-dilaur method DLPE)ethanolamine, efficiency.show oyl-sn-glycero-3-phosphoethanolamine,can behigh utilized Importantly, stability DMPE), to yieldover and we highlongPC:PE also productionincubation findmixtures that times at 4 °C. This comprehensive information would be useful in determining lipid spe- DLPE;mostof(POPC:PCs GVs of(POPC, 1,2-dimyristoyl-sn-glycero-3-phospho withDLPE,these significantly GVs DOPC,DOPC: from DLPE,DMPC), w/o high emulsionand encapsulationPEs DMPC: (1,2-dilaur method DLPE)ethanolamine, efficiency. show oyl-sn-glycero-3-phosphoethanolamine,can behigh utilized Importantly, stabilityDMPE), to yield overand we highPC:PElong also productionincubation mixturesfind that cies and conditions for liposome-based research. (POPC:timesmostofDLPE; GVs ofat 1,2-dimyristoyl-sn-glycero-3-phospho DLPE, withthese4 °C. significantlyThisGVs DOPC: comprehensivefrom DLPE, w/o high emulsionand encapsulation DMPC: informatio method DLPE)ethanolamine,n efficiency. wouldshow can be highbe utilized usefulImportantly, stability DMPE), into determiningyield over and we highlongPC:PE also productionincubation lipidfindmixtures spe-that oftimesciesmost(POPC: GVs and ofat with DLPE,these4conditions °C. significantly ThisGVs DOPC: comprehensive fromfor DLPE,liposome-based w/o high emulsionand encapsulation DMPC:informatio methodresearch. DLPE)n efficiency. wouldshow can be highbe utilized usefulImportantly, stability in to determining yield over we highlong also productionincubation lipidfind spe-that Life 2021, 11, 223 2. 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Solvents, Organic and Other solvents, Reagents including mineral oil (liquid paraffin, fromPOPEpidssn-glycero-3-phosphocholine), Polar,AllCorden (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) the Inc., lipids Pharma Alabaster, (PCs Switzerland and AL. PEs) CalceinDPHPC used LLC, (Solarbio,in Liestal,(1,2-d preparingiphytanoyl-sn-glycero-3-phosphocholine), CH, Beijing, liposomesexcept China) that (Table wasDHPC were used 1) (1,2-diheptanoyl- fromwere as a fluorescent Avantipurchased Li- d = 0.840All g/mL), the lipids were (PCs from and Adamas-Beta PEs) used in Reagent preparing (Shanghai, liposomes China), (Table 1heavy) were mineral purchased oil sn-glycero-3-phosphocholine),pidsdyePOPEfrom to Polar, Corden mark(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) Inc., the Pharma Alabaster,contents Switzerland of AL. GVs. CalceinDPHPC Organic LLC, (Solarbio, Liestal,(1,2-d solvents,iphytanoyl-sn-glycero-3-phosphocholine), CH,Beijing, including except China) that mineral wasDHPC were used oil (1,2-diheptanoyl-from (liquid as a fluorescentAvanti paraffin, Li- fromfrom Sigma–Aldrich Corden Pharma (St. Switzerland Louis, MO, LLC,USA), Liestal, and chloroform CH, except and that methanol DHPC (1,2-diheptanoyl- to dissolve the POPEdyedpidssn-glycero-3-phosphocholine), = 0.840to Polar, (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)mark g/mL), Inc., the Alabaster,contents were from of AL. GVs.Adamas-Beta CalceinDPHPC Organic (Solarbio, (1,2-d soReagentlvents,iphytanoyl-sn-glycero-3-phosphocholine), Beijing, including(Shanghai, China) mineral China), was were used oil heavyfrom (liquid as a fluorescentmineralAvanti paraffin, Li-oil lipidssn-glycero-3-phosphocholine), from Sinopharm Chemical DPHPCReagent (1,2-diphytanoyl-sn-glycero-3-phosphocholine),(Shanghai, China). N-(2 Hydroxyethyl) pipera- pidsdfromdyePOPE = 0.840 toPolar, Sigma–Aldrich mark(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) g/mL), Inc., the Alabaster, contents were (St. from Louis,of AL. GVs.Adamas-Beta Calcein MO, Organic USA), (Solarbio, soReagent anlvents,d chloroform Beijing, including(Shanghai, China) and mineral China), wasmethanol were used oil heavy from (liquidas to a dissolve fluorescentmineralAvanti paraffin, theLi-oil zine-NPOPE′-2-ethanesulfonic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) acid (HEPES), sucrose, glucose, and KOH were were from purchased Avanti Lipidsfrom dyefromlipidsdpids = 0.840to Polar,Sigma–Aldrich frommark g/mL), Inc.,Sinopharm the Alabaster,contents were (St. fromChemical Louis,of AL. GVs. Adamas-Beta Calcein MO, OrganicReagent USA), (Solarbio, so(Shanghai, Reagent anlvents,d chloroform Beijing, including(Shanghai, China). China) and N-(2mineral China), wasmethanol Hydroxyethyl) used oil heavy (liquid as to a dissolve fluorescentmineral paraffin, pipera- theoil Sigma–AldrichPolar, Inc., Alabaster, (St. Louis, AL. MO, Calcein USA). (Solarbio, Beijing, China) was used as a fluorescent dlipidszine-Nfromdye = 0.840to Sigma–Aldrich from ′mark-2-ethanesulfonic g/mL), Sinopharm the contentswere (St. from Chemical Louis,acidof GVs.Adamas-Beta (HEPES), MO, ReagentOrganic USA), sucrose, (Shanghai,soReagent anlvents,d chloroformglucose, (Shanghai,including China). and and N-(2KOHmineral China), methanol Hydroxyethyl) were oil heavy purchased(liquid to dissolvemineral paraffin, pipera- from theoil dye to mark the contents of GVs. Organic solvents, including mineral oil (liquid paraffin, fromzine-NSigma–Aldrichlipidsd = 0.840 Sigma–Aldrich from′-2-ethanesulfonic g/mL), Sinopharm (St. were Louis, (St. fromChemical Louis,acid MO, Adamas-Beta (HEPES), USA).MO, Reagent USA), sucrose, (Shanghai, Reagentand chloroform glucose, (Shanghai, China). and and N-(2KOH China), methanol Hydroxyethyl) were heavy purchased to dissolve mineral pipera- from the oil Tabled = 0.8401. Chemical g/mL), informationwere from on Adamas-Beta all lipids used Reagentto produce (Shanghai, giant vesicles China), (GVs). heavy mineral oil lipidsSigma–Aldrichzine-Nfrom Sigma–Aldrichfrom′-2-ethanesulfonic Sinopharm (St. Louis, (St. Chemical Louis,acid MO, (HEPES), USA). MO, Reagent USA), sucrose, (Shanghai, and chloroformglucose, China). and and N-(2 KOH methanol Hydroxyethyl) were purchased to dissolve pipera- from the Tablefrom 1. Sigma–Aldrich Chemical information (St. Louis,on all lipids MO, us USA),ed to produce and chloroform giant vesicles and (GVs). methanol to dissolve zine-NSigma–AldrichlipidsLipids from′-2-ethanesulfonic Sinopharm (St. Louis,CAS Chemical acid MO, No. (HEPES), USA). Reagent sucrose,Formula (Shanghai, glucose, China). and N-(2KOH StructureHydroxyethyl) were purchased pipera- from Tablethe lipids1. Chemical from information Sinopharm on Chemical all lipids us Reagented to produce (Shanghai, giant vesicles China). (GVs). N-(2 Hydroxyethyl) Sigma–Aldrichzine-NDHPCLipids′-2-ethanesulfonic (1,2-di- 0 (St. Louis,CAS acid MO, No. (HEPES), USA). sucrose,Formula glucose, and KOH Structure were purchased from Tablepiperazine-N 1. Chemical-2-ethanesulfonic information on all acid lipids (HEPES), used to produce sucrose, giant glucose, vesicles and (GVs). KOH were purchased Sigma–AldrichfromDHPCheptanoyl-sn-Lipids Sigma–Aldrich (1,2-di- (St. Louis,CAS (St. Louis,MO, No. USA). MO, USA).Formula Structure Table 1. Chemical information39036-04-9 on all lipidsC us22Hed44 toNO produce8P giant vesicles (GVs). glycero-3-phos-DHPCheptanoyl-sn-Lipids (1,2-di- CAS No. Formula Structure Table 1. Chemical information39036-04-9 on all lipidsC us22Hed44 toNO produce8P giant vesicles (GVs). Table 1.glycero-3-phos-DHPCheptanoyl-sn-ChemicalphocholineLipids (1,2-di- information CAS on all No. lipids used toFormula produce giant vesicles (GVs). Structure 39036-04-9 C22H44NO8P DLPCglycero-3-phos-DHPCheptanoyl-sn-phocholineLipids (1,2-dilau- (1,2-di- CAS No. Formula Structure Lipids CAS No.39036-04-9 FormulaC22H44NO8P Structure DLPCroyl-sn-glycero-glycero-3-phos-heptanoyl-sn-DHPCphocholine (1,2-dilau- (1,2-di- 39036-04-918194-25-7 C2232H4464NO8P DHPC (1,2-diheptanoyl-sn-glycero-3-DLPCglycero-3-phos-royl-sn-glycero-3-phosphocho-heptanoyl-sn-phocholine (1,2-dilau- 39036-04-918194-25-739036-04-9 C CH3222HNO6444NOP 8P phosphocholine DLPCroyl-sn-glycero-glycero-3-phos-3-phosphocho-phocholine line(1,2-dilau- 22 44 8 18194-25-7 C32H64NO8P DLPCroyl-sn-glycero-3-phosphocho-DMPCphocholine (1,2-dilau-line (1,2- 18194-25-7 C32H64NO8P DLPC (1,2-dilauroyl-sn-glycero-3-DLPCroyl-sn-glycero-dimyristoyl-sn-3-phosphocho-DMPC line(1,2-dilau- (1,2- 18194-25-718194-25-718194-24-6 C32CH323664HNO6472NO8P 8P phosphocholine glycero-3-phos-royl-sn-glycero-dimyristoyl-sn-3-phosphocho-DMPCline (1,2- 18194-24-618194-25-7 C3632H7264NO8P glycero-3-phos-dimyristoyl-sn-3-phosphocho-phocholine)DMPCline (1,2- 18194-24-6 C36H72NO8P DMPC (1,2-dimyristoyl-sn-glycero-3-DPPCglycero-3-phos-dimyristoyl-sn-DMPCphocholine) (1,2-dipal-line (1,2- 18194-24-618194-24-6 C36CH3672HNO72NO8P 8P phosphocholine) DPPCdimyristoyl-sn-mitoyl-sn-glyc-glycero-3-phos-phocholine)DMPC (1,2-dipal- (1,2- 18194-24-663-89-8 C3640H7280NO8P DPPCglycero-3-phos-ero-3-phospho-dimyristoyl-sn-mitoyl-sn-glyc-phocholine) (1,2-dipal- 18194-24-663-89-8 C4036H8072NO8P DPPC (1,2-dipalmitoyl-sn-glycero-3-DPPCglycero-3-phos-ero-3-phospho-mitoyl-sn-glyc-phocholine)) (1,2-dipal- Life 2021, 11, x FOR PEER REVIEW 63-89-863-89-8 C40CH4080HNO80NO8P 8P 4 of 16 phosphocholine) DPPCero-3-phospho-mitoyl-sn-glyc-DPHPCphocholine)choline) (1,2-dipal- (1,2- 63-89-8 C40H80NO8P Life 2021, 11, x FOR PEER REVIEW DPPCdiphytanoyl-sn-mitoyl-sn-glyc-ero-3-phospho-DPHPCcholine) (1,2-dipal- (1,2- 4 of 16 207131-40-663-89-8 C4048H8096NO8P DPHPC (1,2-diphytanoyl-sn-glycero-3-diphytanoyl-sn-glycero-3-phos-ero-3-phospho-mitoyl-sn-glyc-DPHPCcholine) (1,2- Life 2021, 11, x FOR PEER REVIEW 207131-40-6 C H48 NO96 P 8 4 of 16 DOPC (1,2-di- 207131-40-663-89-8 48C4096H80NO8 8P phosphocholine) diphytanoyl-sn-glycero-3-phos-ero-3-phospho-DPHPCphocholine)choline) (1,2- 207131-40-6 C48H96NO8P Life 2021, 11, x FOR PEER REVIEW glycero-3-phos-oleoyl-sn-glyc-phocholine) 4 of 16 diphytanoyl-sn-DSPC(1,2-dis-DOPCDPHPCcholine) (1,2-di- (1,2- 4235-95-4 C44H84NO8P ero-3-phospho- 207131-40-6 C48H96NO8P DSPC(1,2-distearoyl-sn-glycero-3-diphytanoyl-sn-tearoyl-sn-glyc-glycero-3-phos-oleoyl-sn-glyc-DSPC(1,2-dis-DPHPCphocholine) (1,2- DOPC (1,2-di-816-94-4816-94-4 C CH4844HNO9688NOP 8P phosphocholine) choline) 207131-40-64235-95-4 44C4488H84NO8 8P glycero-3-phos-diphytanoyl-sn-ero-3-phospho-tearoyl-sn-glyc-DSPC(1,2-dis-phocholine) oleoyl-sn-glyc-POPC (1-Pal- 207131-40-6816-94-4 C4448H8896NO8P tearoyl-sn-glyc-glycero-3-phos-ero-3-phospho-DOPCDSPC(1,2-dis-phocholine)choline) (1,2-di- 4235-95-4 C44H84NO8P ero-3-phospho-choline) 816-94-4 C44H88NO8P DOPC (1,2-dioleoyl-sn-glycero-3-mitoyl-2-oleoyl-ero-3-phospho-oleoyl-sn-glyc-choline) tearoyl-sn-glyc-DSPC(1,2-dis-POPCphocholine) (1-Pal- 26853-31-6 C42H82NO8P choline) 4235-95-44235-95-4 C44CH4484HNO8488NO8P 8P phosphocholine) ero-3-phospho-sn-glycero-3- 816-94-4 C H NO P mitoyl-2-oleoyl-tearoyl-sn-glyc-ero-3-phospho-DSPC(1,2-dis-choline) POPC (1-Pal- 44 88 8 phosphocholine)choline) 26853-31-6816-94-4 C42H82NO8P ero-3-phospho-tearoyl-sn-glyc-sn-glycero-3-choline) POPC (1-Palmitoyl-2-oleoyl-sn-glycero-DLPEmitoyl-2-oleoyl- (1,2-dilau- 816-94-4 C44H88NO8P ero-3-phospho-POPCcholine) (1-Pal- 26853-31-626853-31-6 C CH42HNO82NOP 8P 3-phosphocholine) phosphocholine)sn-glycero-3- 42 82 8 royl-sn-glycero-mitoyl-2-oleoyl-choline) DLPEphosphocholine) (1,2-dilau- 59752-57-726853-31-6 C2942H5882NO88P 3-phosphoethan-sn-glycero-3- DLPE (1,2-dilauroyl-sn-glycero-3-DLPEroyl-sn-glycero- (1,2-dilau- phosphocholine)olamine) 59752-57-7 59752-57-7 C29CH2958HNO58NO8P 8P phosphoethanolamine)3-phosphoethan- royl-sn-glycero-DMPE (1,2- DLPE (1,2-dilau- 59752-57-7 C29H58NO8P 3-phosphoethan-olamine) royl-sn-glycero-dimyristoyl-sn- DMPE (1,2-dimyristoyl-sn-glycero-3-DMPEolamine) (1,2- 59752-57-7 C29H58NO8P 3-phosphoethan-glycero-3-phos-998-07-2998-07-2 C33CH3366HNO66NO8P 8P phosphoethanolamine) dimyristoyl-sn- phoethanola-DMPEolamine) (1,2- glycero-3-phos- 998-07-2 C33H66NO8P dimyristoyl-sn-mine) phoethanola-DMPE (1,2- glycero-3-phos- 998-07-2 C33H66NO8P DPPEdimyristoyl-sn- (1,2-dipal- phoethanola-mine) glycero-3-phos-mitoyl-sn-gly- 998-07-2 C33H66NO8P DPPEmine) (1,2-dipal- 923-61-5 C37H74NO8P cero-3-phospho-phoethanola- DPPEmitoyl-sn-gly- (1,2-dipal- ethanolaminemine) 923-61-5 C37H74NO8P cero-3-phospho- DSPEmitoyl-sn-gly- (1,2-dis- DPPE (1,2-dipal- 923-61-5 C37H74NO8P cero-3-phospho-ethanolamine tearoyl-sn-glyc-mitoyl-sn-gly- DSPEethanolamine (1,2-dis- 923-61-5 C37H74NO8P cero-3-phospho-ero-3-phos- 1069-79-0 C41H82NO8P tearoyl-sn-glyc- DSPEethanolaminephoethanola- (1,2-dis- ero-3-phos- 1069-79-0 C41H82NO8P tearoyl-sn-glyc-mine) DSPEphoethanola- (1,2-dis- ero-3-phos- 1069-79-0 C41H82NO8P tearoyl-sn-glyc-DOPE (1,2-di- phoethanola-mine) oleoyl-sn-glyc-ero-3-phos- 1069-79-0 C41H82NO8P DOPEmine) (1,2-di- phoethanola-ero-3-phos- 4004-05-1 C41H78NO8P oleoyl-sn-glyc- DOPEphoethanola-mine) (1,2-di- ero-3-phos- 4004-05-1 C41H78NO8P oleoyl-sn-glyc-mine) DOPEphoethanola- (1,2-di- ero-3-phos- 4004-05-1 C41H78NO8P oleoyl-sn-glyc-POPE (1-pal- phoethanola-mine) mitoyl-2-oleoyl-ero-3-phos- 4004-05-1 C41H78NO8P POPEmine) (1-pal- sn-glycero-3-phoethanola- 26662-94-2 C39H76NO8P mitoyl-2-oleoyl- phosphoethano-POPEmine) (1-pal- sn-glycero-3- 26662-94-2 C39H76NO8P mitoyl-2-oleoyl-lamine) phosphoethano-POPE (1-pal- sn-glycero-3- 26662-94-2 C39H76NO8P mitoyl-2-oleoyl- 2.2.phosphoethano- Solubilizedlamine) Lipids, Inner and Bottom Aqueous Solutions Preparation sn-glycero-3- 26662-94-2 C39H76NO8P Alllamine) the PC lipids including POPC, DOPC, DMPC, DPPC (1,2-dipalmitoyl-sn-glycero- 2.2.phosphoethano- Solubilized Lipids, Inner and Bottom Aqueous Solutions Preparation 3-phosphocholine),lamine) DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DHPC (1,2-di- heptanoyl-sn-glycero-3-phosphocholine),2.2. SolubilizedAll the PC Lipids, lipids Innerincluding and Bo POPC,ttom Aqueous DOPC, DLPC DMPC,Solutions (1,2-dilauroyl-sn-glycero-3-phosphocho- DPPC Preparation (1,2-dipalmitoyl-sn-glycero- 3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DHPC (1,2-di- line),2.2. SolubilizedAll and the DPHPC PC Lipids, lipids (1,2-diphytanoyl-sn-glycero-3-phosphocholine) Innerincluding and Bo POPC,ttom Aqueous DOPC, DMPC,Solutions DPPC Preparation (1,2-dipalmitoyl-sn-glycero- were dissolved in chlo- roformheptanoyl-sn-glycero-3-phosphocholine),3-phosphocholine), to make 100 mg/mLDSPC (1,2-distearoyl-snstock solutions. DLPC Th-glycero-3-phosphocholine),e stock (1,2-dilauroyl-sn-glycero-3-phosphocho- solutions of PE, including DHPC POPE (1,2-di- (1- All the PC lipids including POPC, DOPC, DMPC, DPPC (1,2-dipalmitoyl-sn-glycero- palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine)line),heptanoyl-sn-glycero-3-phosphocholine), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) DLPC (1,2-dilauroyl-sn-glycero-3-phosphocho- and DOPE were (1,2-dioleoyl-sn-glyc- dissolved in chlo- 3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DHPC (1,2-di- ero-3-phosphoethanolamine),roformline), and to DPHPCmake 100 (1,2-diphytanoyl-sn-glycero-3-phosphocholine) mg/mL stock were solutions. mixed Th 50e stock mg/mL solutions in chloroform. of PE,were including dissolved DMPE POPE in chlo-(1,2- (1- heptanoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocho- dimyristoyl-sn-glycero-3-phosphoethanolamine),palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine)roform to make 100 mg/mL stock solutions. The stock DLPE solutions and DOPE(1,2-dilauroyl-sn-glycero-3- of PE, (1,2-dioleoyl-sn-glyc- including POPE (1- line), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) were dissolved in chlo- phosphoethanolamine),ero-3-phosphoethanolamine),palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine) DPPE were (1,2-dipalmito mixed 50 yl-sn-glycero-3-phosmg/mL and in DOPEchloroform. (1,2-dioleoyl-sn-glyc-pho-ethanolamine), DMPE (1,2- roform to make 100 mg/mL stock solutions. The stock solutions of PE, including POPE (1- anddimyristoyl-sn-glycero-3-phosphoethanolamine),ero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) were mixed 50 mg/mLDLPE in (1,2-dilauroyl-sn-glycero-3-chloroform. were dissolvedDMPE (1,2-in phosphoethanolamine),palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine) DPPE (1,2-dipalmitoyl-sn-glycero-3-phos and DOPE (1,2-dioleoyl-sn-glyc-pho-ethanolamine), CHCldimyristoyl-sn-glycero-3-phosphoethanolamine),3/MeOH/ddH2O (65:35:8). The final stock soluti DLPEon concentration (1,2-dilauroyl-sn-glycero-3- of DMPE, DLPE, ero-3-phosphoethanolamine), were mixed 50 mg/mL in chloroform. DMPE (1,2- andphosphoethanolamine), DPPEDSPE was (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) 15 mg/mL, DPPEand 5 mg/mL(1,2-dipalmito for DSPE.yl-sn-glycero-3-phos The stock solutions werepho-ethanolamine), were dissolved used to gen- in dimyristoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-sn-glycero-3- erateCHCland lipid–carrying3DSPE/MeOH/ddH (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)2 Ooil (65:35:8). solutions The and final filled stock with soluti nitrogenon concentration for storage. were Theof DMPE, dissolvedlipids POPC,DLPE, in andphosphoethanolamine), DPPE was 15 mg/mL, DPPEand 5 mg/mL(1,2-dipalmito for DSPE.yl-sn-glycero-3-phos The stock solutionspho-ethanolamine), were used to gen- CHCl3/MeOH/ddH2O (65:35:8). The final stock solution concentration of DMPE, DLPE, and DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) were dissolved in erateand DPPE lipid–carrying was 15 mg/mL, oil solutions and 5 mg/mL and filled for wiDSPE.th nitrogen The stock for solutions storage. wereThe lipids used toPOPC, gen- CHCl3/MeOH/ddH2O (65:35:8). The final stock solution concentration of DMPE, DLPE, erate lipid–carrying oil solutions and filled with nitrogen for storage. The lipids POPC, and DPPE was 15 mg/mL, and 5 mg/mL for DSPE. The stock solutions were used to gen- erate lipid–carrying oil solutions and filled with nitrogen for storage. The lipids POPC,

Life 2021, 11, x FOR PEER REVIEW 4 of 16

Life 2021, 11, x FOR PEER REVIEW 4 of 16

DOPC (1,2-di- oleoyl-sn-glyc- Life 2021, 11, x FOR PEER REVIEW 4235-95-4 C44H84NO8P 4 of 16 ero-3-phospho-DOPC (1,2-di- Life 2021, 11, x FOR PEER REVIEW oleoyl-sn-glyc-choline) 4 of 16 4235-95-4 C44H84NO8P ero-3-phospho-POPC (1-Pal- DOPC (1,2-di- mitoyl-2-oleoyl-choline) oleoyl-sn-glyc- 26853-31-6 C42H82NO8P DOPCPOPCsn-glycero-3- (1,2-di-(1-Pal- 4235-95-4 C44H84NO8P ero-3-phospho- phosphocholine)mitoyl-2-oleoyl-oleoyl-sn-glyc- choline) 26853-31-64235-95-4 C4244H8284NO8P DLPEsn-glycero-3- (1,2-dilau- ero-3-phospho-POPC (1-Pal- phosphocholine)royl-sn-glycero- mitoyl-2-oleoyl-choline) 59752-57-7 C29H58NO8P 3-phosphoethan-DLPEPOPC (1,2-dilau- (1-Pal- 26853-31-6 C42H82NO8P sn-glycero-3- royl-sn-glycero-mitoyl-2-oleoyl-olamine) phosphocholine) 59752-57-726853-31-6 C2942H5882NO8P 3-phosphoethan-DMPE (1,2- DLPEsn-glycero-3- (1,2-dilau- dimyristoyl-sn-olamine) phosphocholine)royl-sn-glycero- DLPEglycero-3-phos-DMPE (1,2-dilau- (1,2- 59752-57-7998-07-2 CC2933HH5866NONO88PP 3-phosphoethan- royl-sn-glycero-dimyristoyl-sn-phoethanola- olamine) 59752-57-7 C29H58NO8P glycero-3-phos-mine) 998-07-2 C33H66NO8P 3-phosphoethan-DMPE (1,2- DPPEphoethanola- (1,2-dipal- dimyristoyl-sn-olamine) Life 2021, 11, 223 mitoyl-sn-gly-mine) 4 of 13 glycero-3-phos-DMPE (1,2- 998-07-2923-61-5 CC3337HH6674NONO88PP cero-3-phospho- DPPEdimyristoyl-sn-phoethanola- (1,2-dipal- ethanolamine glycero-3-phos-mitoyl-sn-gly- 998-07-2 C33H66NO8P mine) 923-61-5 C37H74NO8P cero-3-phospho-DSPE (1,2-dis- Table 1. Cont. DPPEphoethanola- (1,2-dipal- tearoyl-sn-glyc-ethanolaminemine) Lipidsmitoyl-sn-gly- CAS No. Formula Structure ero-3-phos- 1069-79-0923-61-5 CC3741HH7482NONO88PP cero-3-phospho-DPPEDSPE (1,2-dipal- (1,2-dis- phoethanola- DPPE (1,2-dipalmitoyl-sn-glycero-3-tearoyl-sn-glyc-ethanolaminemitoyl-sn-gly- 923-61-5923-61-5 C37CH3774HNO74NO8P 8P phospho-ethanolamine ero-3-phos-mine) 1069-79-0 C41H82NO8P cero-3-phospho-DSPE (1,2-dis- DOPEphoethanola- (1,2-di- tearoyl-sn-glyc-ethanolamine DSPE (1,2-distearoyl-sn-glycero-3-oleoyl-sn-glyc-DSPEmine) (1,2-dis- ero-3-phos- 1069-79-01069-79-0 C41CH4182HNO82NO8P 8P phosphoethanolamine) ero-3-phos- 4004-05-1 C41H78NO8P tearoyl-sn-glyc-DOPEphoethanola- (1,2-di- phoethanola- oleoyl-sn-glyc-ero-3-phos- 1069-79-0 C41H82NO8P DOPE (1,2-dioleoyl-sn-glycero-3- mine) ero-3-phos-mine) 4004-05-14004-05-1 C CH41HNO78NOP 8P phosphoethanolamine) DOPEphoethanola- (1,2-di- 41 78 8 phoethanola-POPE (1-pal- oleoyl-sn-glyc-mine) mitoyl-2-oleoyl-mine) POPE (1-palmitoyl-2-oleoyl-sn-glycero-DOPEero-3-phos- (1,2-di- 4004-05-1 C41H78NO8P sn-glycero-3-26662-94-226662-94-2 C39CH3976HNO76NO8P 8P 3-phosphoethanolamine)oleoyl-sn-glyc-phoethanola-POPE (1-pal- phosphoethano- mitoyl-2-oleoyl-ero-3-phos-mine) 4004-05-1 C41H78NO8P sn-glycero-3-lamine) 26662-94-2 C39H76NO8P POPEphoethanola- (1-pal- phosphoethano- mitoyl-2-oleoyl-2.2. Solubilizedmine) Lipids, Inner and Bottom Aqueous Solutions Preparation 2.2. Solubilizedlamine) Lipids, Inner and Bottom Aqueous Solutions Preparation sn-glycero-3-POPEAll (1-pal- the PC lipids26662-94-2 including POPC,C39 DOPC,H76NO DMPC,8P DPPC (1,2-dipalmitoyl-sn-glycero-3- All the PC lipids including POPC, DOPC, DMPC, DPPC (1,2-dipalmitoyl-sn-glycero- phosphoethano-mitoyl-2-oleoyl-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DHPC (1,2-diheptanoyl - 2.2.3-phosphocholine), Solubilized Lipids, DSPC Inner and(1,2-distearoyl-sn Bottom Aqueous-glycero-3-phosphocholine), Solutions Preparation DHPC (1,2-di- sn-glycero-3-phosphocholine),sn-glycero-3-lamine) 26662-94-2 DLPCC (1,2-dilauroyl-sn-glycero-3-phosphocholine),39H76NO8P and heptanoyl-sn-glycero-3-phosphocholine), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocho- phosphoethano-DPHPCAll the (1,2-diphytanoyl-sn-glycero-3-phosphocholine) PC lipids including POPC, DOPC, DMPC, DPPC were (1,2-dipalmitoyl-sn-glycero- dissolved in chloroform to line), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) were dissolved in chlo- 2.2.3-phosphocholine),make Solubilizedlamine) 100 mg/mL Lipids, stockDSPC Inner solutions. and(1,2-distearoyl-sn Bottom The Aqueous stock-glycero-3-phosphocholine), solutions Solutions of Preparation PE, including POPE DHPC (1-palmitoyl- (1,2-di- heptanoyl-sn-glycero-3-phosphocholine),roform2-oleoyl-sn-glycero-3-phospho-ethanolamine) to make 100 mg/mL stock solutions. DLPC The and stock(1,2-dilauroyl-sn-glycero-3-phosphocho- DOPE solutions (1,2-dioleoyl-sn-glycero-3- of PE, including POPE phos- (1- All the PC lipids including POPC, DOPC, DMPC, DPPC (1,2-dipalmitoyl-sn-glycero- line),palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine)phoethanolamine), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) were mixed 50 mg/mL in chloroform. and DOPE DMPE were (1,2-dioleoyl-sn-glyc- (1,2-dimyristoyl-sn- dissolved in chlo- 3-phosphocholine),2.2. Solubilized Lipids, DSPC Inner and(1,2-distearoyl-sn Bottom Aqueous-glycero-3-phosphocholine), Solutions Preparation DHPC (1,2-di- roformero-3-phosphoethanolamine),glycero-3-phosphoethanolamine), to make 100 mg/mL stock were solutions. DLPE mixed (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), Th 50e stock mg/mL solutions in chloroform. of PE, including DMPE POPE (1,2- (1- heptanoyl-sn-glycero-3-phosphocholine),All the PC lipids including POPC, DOPC, DLPC DMPC, (1,2-dilauroyl-sn-glycero-3-phosphocho- DPPC (1,2-dipalmitoyl-sn-glycero- palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine)dimyristoyl-sn-glycero-3-phosphoethanolamine),DPPE (1,2-dipalmitoyl-sn-glycero-3-phospho-ethanolamine), DLPE and DOPE(1,2-dilauroyl-sn-glycero-3- and (1,2-dioleoyl-sn-glyc- DSPE (1,2-distearoyl- line),3-phosphocholine), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), were dissolved DHPC in(1,2-di- chlo- ero-3-phosphoethanolamine),phosphoethanolamine),sn-glycero-3-phosphoethanolamine) DPPE were (1,2-dipalmito mixed were dissolved 50 yl-sn-glycero-3-phosmg/mL in CHCl in chloroform./MeOH/ddHpho-ethanolamine), DMPEO (65:35:8). (1,2- roformheptanoyl-sn-glycero-3-phosphocholine), to make 100 mg/mL stock solutions. DLPC The stock (1,2-dilauroyl-sn-glycero-3-phosphocho- solutions3 of PE, including2 POPE (1- dimyristoyl-sn-glycero-3-phosphoethanolamine),andThe finalDSPE stock (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) solution concentration of DMPE, DLPE,DLPE and (1,2-dilauroyl-sn-glycero-3- DPPE were was 15dissolved mg/mL, andin palmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine)line), and DPHPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) and DOPE were (1,2-dioleoyl-sn-glyc- dissolved in chlo- phosphoethanolamine),CHCl5 mg/mL3/MeOH/ddH for DSPE.2O The(65:35:8). DPPE stock The solutions(1,2-dipalmito final stock were solutiyl-sn-glycero-3-phos usedon to generateconcentration lipid–carryingpho-ethanolamine), of DMPE, oilDLPE, solu- ero-3-phosphoethanolamine),roform to make 100 mg/mL stock were solutions. mixed Th 50e stockmg/mL solutions in chloroform. of PE, including DMPE POPE (1,2- (1- andtions DPPE and was filled 15 with mg/mL, nitrogen and for5 mg/mL storage. for The DSPE. lipids The POPC, stock DMPC,solutions DPPC, were DSPC,used to DLPE, gen- andpalmitoyl-2-oleoyl-sn-glycero-3-phospho-ethanolamine) DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) and DOPE (1,2-dioleoyl-sn-glyc-were dissolved in dimyristoyl-sn-glycero-3-phosphoethanolamine),erateDOPE, lipid–carrying POPE, DPPE, oil and solutions DSPE stockand filled solutions with were nitrogenDLPE mixed for with(1,2-dilauroyl-sn-glycero-3- storage. mineral The oil lipids to produce POPC, a CHClero-3-phosphoethanolamine),3/MeOH/ddH2O (65:35:8). were The finalmixed stock 50 soluti mg/mLon concentration in chloroform. of DMPE, DMPE DLPE, (1,2- phosphoethanolamine),0.1 mg/mL lipid–oil solution. DPPE Heavy (1,2-dipalmito mineral oilyl-sn-glycero-3-phos was used to preparepho-ethanolamine), 0.1 mg/mL lipid–oil anddimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE was 15 mg/mL, and 5 mg/mL for DSPE. TheDLPE stock solutions(1,2-dilauroyl-sn-glycero-3- were used to gen- andsolutions DSPE of DOPC,(1,2-distearoyl-sn-glycero-3-phosphoethanolamine) DHPC, DLPC, DPHPC, and DMPE (Table2). Finally,were thedissolved chloroform in eratephosphoethanolamine), lipid–carrying oil solutionsDPPE and(1,2-dipalmito filled withyl-sn-glycero-3-phos nitrogen for storage.pho-ethanolamine), The lipids POPC, CHClwas3 evaporated/MeOH/ddH by2O heating (65:35:8). the The lipid-oil final solutions stock soluti in anon openconcentration tube at 80 of◦C DMPE, for 30 min.DLPE, andand DPPEDSPE was (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) 15 mg/mL, and 5 mg/mL for DSPE. The stock solutions were were dissolved used to gen- in CHCl3/MeOH/ddH2O (65:35:8). The final stock solution concentration of DMPE, DLPE, erateTable lipid–carrying 2. Sonication conditions oil solutions for emulsion and filled produced with nitrogen with different for storage. lipids. The lipids POPC, and DPPE was 15 mg/mL, and 5 mg/mL for DSPE. The stock solutions were used to gen- erate lipid–carryingLipids oil solutions Oil and filled with nitrogen Sonication for storage. Conditions The lipids POPC, DHPC hMO 1 sonication on ice, equilibration on ice DLPC hMO sonication on ice, equilibration on ice DMPC MO 2 sonication at RT, equilibration at RT DPPC hMO sonication on ice, equilibration at RT DPHPC hMO sonication on ice, equilibration on ice DSPC MO sonication at RT, equilibration at RT DOPC hMO sonication on ice, equilibration at RT POPC MO sonication on ice, equilibration on ice DLPE MO sonication on ice, equilibration at RT DMPE hMO sonication on ice, equilibration at RT DPPE hMO sonication on ice, equilibration at RT DSPE MO sonication on ice, equilibration at RT DOPE hMO sonication on ice, equilibration on ice POPE hMO sonication on ice, equilibration at RT POPC: DLPE MO sonication on ice, equilibration on ice DOPC: DLPE hMO sonication on ice, equilibration at RT DMPC: DLPE MO sonication at RT, equilibration at RT 1 heavy mineral oil, 2 mineral oil. Life 2021, 11, 223 5 of 13

Two aqueous solutions, inner and bottom aqueous solutions, were made using ddH2O water. The inner aqueous solution contained 50 mM HEPES-KOH (pH 7.6), 500 mM sucrose, 0.2 mM calcein. The bottom aqueous solution was also prepared with 50 mM HEPES-KOH (pH 7.6) and 500 mM glucose to maintain the isosmotic conditions across the lipid bilayer membranes.

2.3. Preparation of Calcein-Stained Vesicles by the Inverted Emulsion Method The w/o emulsion transfer method was chosen to produce GVs as previously de- scribed [15] with some minor modifications for some lipids. Calcein was added to the inner aqueous solution enabling quantitative analysis of GVs by flow cytometry. After the lipids were dissolved and the chloroform was entirely evaporated, the w/o emulsion was made by sonication using an ultrasonic processor (SONICS & MATERIALS, INC, VCX750, Newton, Connecticut, USA) on ice for 1 min (Ampl. 40%, pulse on/off 2 s/1 s) and equilibrated on ice for 10 min. Considering that lipid species used in this study have different structures and transition temperatures, the sonication conditions for the emulsion produced with those lipids were optimized, as listed in Table2. The bottom aqueous solution was placed in a new microcentrifuge tube, and the w/o emulsion obtained in the previous step was gently poured on top of the bottom aqueous solution. After standing for 5−10 min to equilibrate the interface, the emulsion was centrifuged to form a pellet of GVs. The supernatant oil was removed and a fresh bottom aqueous solution was added to resuspend the pellet. Oil traces were removed by another centrifugation round. Finally, 100 µL of this GV suspension was prepared and immediately analyzed by IFC or stored at 4 ◦C for further stability analysis.

2.4. GV Analysis by Imaging Flow Cytometry (IFC) Like Ishiodori et al. [15], we used the ImageStream Mark II system and INSPIRE acquisition software (Amnis/Millipore, Seattle, Washington, USA) to measure GVs based on the fluorescence intensities from encapsulated calcein. The emission from calcein (488 nm) was detected in Channel 2 with a 505−560 nm filter; bright field and side scatter (SSC) data were collected in Channel 4 and Channel 6 (785 nm), respectively. All samples were acquired with 60× magnification with a setup of low flow rate/high sensitivity. 50,000 events were recorded for each GV composition and the measurement data was analyzed by IDEAS analysis software (Amnis/Millipore, Seattle, Washington, USA). In this type of flow cytometry, internal size standard beads are run concurrently and used for daily calibration as well as real-time velocity detection and autofocus.

2.5. Confocal Microscopy Observation The produced GVs were dropped (8 µL) on a microscope slide with a silicon imaging spacer (1 well, diam. × thickness 9 mm × 0.12 mm, Sigma–Aldrich, St. Louis, MO, USA), which were ultra-thin adhesive spacers peeled and stuck to slides to confine specimens without compression. The sample was then covered with a coverslip and left to stand for 10 min to allow the sediment of vesicles to travel to the bottom of the chamber by gravity. GV samples were observed with a Ti2-E inverted microscope (Nikon Ti2-E, Yokohama, Japan), and images were acquired using a confocal microscope (Nikon C2plus, Yokohama, Japan). The green fluorescence of calcein inside the GVs was excited by using a 488 nm laser with emission collected at 498–535 nm. Image analysis was performed using NIS- ELEMENTS C-ER software (Nikon, Yokohama, Japan).

2.6. Stability Analysis GVs produced with POPC, DOPC, DLPE, DOPC: DLPE (7:3) mixture, and POPC: DLPE (6:4) mixture were stored at 4 ◦C for up to one month. We investigated the con- centration and size distribution of the resulting GVs via IFC at indicated time points, 0 h, 6 h, 12 h, 24 h, 3 days, 1 week, 2 weeks, and 1 month. We also directly observed these GV samples under the confocal microscope (Nikon C2plus, Yokohama, Japan). Life 2021, 11, 223 6 of 13

3. Results 3.1. Using IFC to Analyze GVs Produced by the w/o Emulsion Transfer Method The experimental procedures are summarized in Figure1a. An advantage of using IFC to characterize GVs was that the size distribution of all GVs could be clearly displayed and recorded. We employed IFC and microscopy to analyze the properties of GVs produced from different lipid components. As vesicles produced by the inverted w/o emulsion trans- fer method are of high encapsulation efficiency and mainly in the uni-lamellar form [15,16],

Life 2021, 11, x FOR PEER REVIEWwe chose this method to prepare GVs with PCs and PEs with or without calcein8 of 16 encap- sulations [15]. The obtained GVs were then subjected to IFC analysis as described in the “Materials and Methods” Section. a Produce GVs with PCs/PEs

Analysis in IFC and confocal microscopy

Concentration Size distrubution

Stability after storage in refrigerator b

POPCDMPC DOPC DPPC DSPC

10 µm

DLPE DOPE POPE DMPE DPHPC

cd9×109 9×109 GVs GVs 9 9 8×10 GVs with calcein 8×10 GVs with calcein ) ) 1 1 9 9 – – 7×10 7×10

6×109 6×109

5×109 5×109

4×109 4×109

3×109 3×109

2×109 2×109 GV Concentration (mL GV Concentration (mL GV Concentration

1×109 1×109

0 0 POPC DMPC DOPC DPPC DSPC DPHPC DLPC DLPE DOPE DPPE DSPE POPE DMPE PCs PEs

Figure 1. GVsFigure produced 1. GVs produced with different with different phosphatidylcholines phosphatidylcholines (PCs) (PCs) andand phosphatidylethanolamines (Pes). (Pes). (a) The ( aresearch) The research roadmap ofroadmap this study, of this (b study,) The ( fluorescenceb) The fluorescence microscope microscope images images of of GVs GVs preparedprepared with with PCs PCs and and PEs. PEs. Scale Scale bar = 10 bar μm. = 10Theµ m. The concentrationconcentration of GVs made of GVs with made different with different PCs PCs (c,d )(c PEs.,d) PEs. 3.2. Comparing GVs Prepared with Different PC, PE, and PC:DLPE Mixture

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Following the procedure developed previously [15], we further made some fine-tuning for each kind of GV sample. Based on a sequence diagram for various SSC features versus brightfield, we could separate target GVs from the internal calibration beads and other byproducts such as oil droplets. Finally, we could also obtain characteristics of target GVs such as purity, calcein encapsulation efficiency, and vesicle size from the IFC analysis results as listed in Table3 and Table S1. All the data were summarized from at least three independent replications, showing low standard deviations in each sample and outstanding reproducibility via IFC-based analysis.

Table 3. Comparison of high yield of vesicles *.

Purity Concentration Mean Diameter Encapsulation Efficiency GVs (%) (Objects/mL) (µm) (%) POPC 92.45 ± 0.30 5.14 ± 0.22 ×109 4.08 ± 0.15 91.07 ± 0.15 DOPC 96.85 ± 0.13 6.54 ± 0.17 ×109 4.09 ± 0.05 94.12 ± 0.30 DMPC 93.33 ± 0.21 2.94 ± 0.13 ×109 4.69 ± 0.06 89.24 ± 0.35 DLPE 97.01 ± 0.71 7.02 ± 0.90 ×109 3.66 ± 0.29 91.61 ± 0.35 POPC: DLPE_6:4 97.64 ± 0.09 6.68 ± 0.78 ×109 3.75 ± 0.06 93.07 ± 0.04 DOPC: DLPE_7:3 93.10 ± 0.32 6.94 ± 0.60 ×109 3.76 ± 0.10 94.21 ± 0.37 DMPC: DLPE_6:4 91.61 ± 0.32 5.68 ± 0.14 ×109 3.44 ± 0.02 96.03 ± 0.05 * represents means ± standard error, Sample size (n) = 3.

3.2. Comparing GVs Prepared with Different PC, PE, and PC:DLPE Mixture To investigate the effects of PC and PE species on vesicle formation, we firstly prepared GVs with seven kinds of PCs (POPC, DMPC, DOPC, DPPC, DSPC, DPHPC, and DLPC) or six kinds of PEs (DLPE, DOPE, DPPE, DSPE, POPE, and DMPE). Before IFC and confocal observation, generated GVs were viewed under a fluorescence microscope as soon as they were prepared. As shown in Figure1b (upper panel), the fluorescence microscope showed that most PCs successfully produced GVs, except for DHPC and DLPC which yielded inferior productions (Figure S1). For GVs prepared with PEs, DLPE obtained a relatively higher yield of GVs as compared with DOPE, POPE, and DMPE species (Figure1b, lower panel). However, the fluorescence microscope could barely detect GV products with DSPE (data not shown). The size of DLPE-derived vesicles seems smaller than DMPC-derived ones as judged from the fluorescence microscopy. Then we investigated all the GV samples via IFC, with which we calculated the con- centration of produced GVs. As plotted in Figure1c, POPC, DMPC, and DOPC had higher yields of GVs than DPPC, DSPC, DPHPC, and DLPC. Overall, DOPC could achieve the highest vesicle production (6.54 ± 0.17 × 109 vesicles/mL) and calcein encapsulation efficiency (94.12 ± 0.30%, Table3). Out of 13 kinds of lipids tested in this study, the concen- tration of GVs from DLPE reaches the highest at 7.02 ± 0.90 × 109 vesicles/mL (Figure1d and Table3). These results acquired from IFC were mainly consistent with the microscopy images. Moreover, with IFC we could compare detailed analysis among samples, and we conclude that POPC, DMPC, DOPC, and DLPE allow for efficient GV production. According to the above concentration results, we tried to prepare GVs with mixed POPC, DOPC, and DMPC with DLPE in ratios of 6:4, 7:3, 8:2, and 9:1, because it was reported that the PC/PE ratio is a critical modulator of membrane integrity [30,41]. For GVs with POPC: DLPE mixture, the yields of vesicles suggested that the extra DLPE could enhance the vesicle formation at a ratio of around 6:4 and 7:3, which is compa- rable to the concentration with pure DLPE (Figure2a). We obtained the highest yield vesicle product of 6.68 ± 0.78 × 109 vesicles/mL in the ratio of 6:4 (Table3). When DOPC was mixed with DLPE in a ratio of 7:3, the mixture reached the highest yields of 6.94 ± 0.60 × 109 vesicles/mL, which was a little higher than the mix in the ratio of 8:2 (Figure2a). Once DMPC mixed with DLPE in the proportion 6:4, the concentration of produced GV (5.68 ± 0.14 × 109 vesicles/mL) was obviously higher than GVs in the ratio of 7:3, 8:2, and 9:1 (Figure2a). The fluorescence microscope showed an apparent Life 2021, 11, 223 8 of 13

Life 2021, 11, x FOR PEER REVIEW 10 of 16

difference in the concentration of vesicles among POPC: DLPE-, DOPC: DLPE-, and DMPC: DLPE-derived GVs (Figure2b and Figure S2), which is in accordance with IFC results. a 9×109 GVs 8×109 GVs with calcein

) 9 1 7×10 –

6×109

5×109

4×109

3×109

2×109 GV Concentration (mL

1×109

0 POPCDMPCDOPC DLPE 6:4 7:3 8:2 9:1 6:4 7:3 8:2 9:1 6:4 7:3 8:2 9:1 POPC:DLPE DMPC:DLPE DOPC:DLPE b POPC:DLPE (6:4) DMPC:DLPE (6:4) DOPC:DLPE (7:3)

10 µm

Figure 2. GVsFigure produced 2. GVs produced with different with different ratio of ratio PC: of DLPE PC: DLPE mixture. mixture. (a) The(a) The concentration concentration of of GVs GVs prepared prepared withwith POPC: DLPE, DLPE, DMPC: DLPE, and DOPC: DLPE, (b) The fluorescence microscope images of GVs prepared with PCs: DLPE. Scale DMPC: DLPE, and DOPC: DLPE, (b) The fluorescence microscope images of GVs prepared with PCs: DLPE. Scale bar = 10 µm. bar = 10 μm.

3.3. Size3.3. Distribution-BasedSize Distribution-based Stability Stability Analysis of of GVs GVs via viaIFC IFC SinceSince IFC IFC can can measure measure the the size size ofof single particles, particles, we wecompared compared size distributions size distributions amongamong GV GV composition composition with with higher higher yields: yields: POPC, POPC, DOPC, DOPC, DLPE, DOPC: DLPE, DLPE DOPC: (7:3), DLPE and (7:3), POPC:DLPE (6:4). For every sample, 50,000 particles were registered regardless of the and POPC:DLPE (6:4). For every sample, 50,000 particles were registered regardless of concentrations. Purity and encapsulation efficiency were defined as the ratio of GVs the concentrations.excluding beads Purityand other and byproducts encapsulation such as oil efficiency droplets in were all obtained defined objects as the and ratio the of GVs excludingratio of beads GVs containing and other calcein byproducts over all suchGVs, asrespectively. oil droplets As listed in all in obtained Table 3, the objects mean and the ratio ofsizes GVs of containingPOPC and DOPC calcein vesicles over all were GVs, almost respectively. identical As(~4.1 listed μm), inwhich Table are3, theslightly mean sizes of POPCsmaller and than DOPC DMPC vesicles (~4.7 μm). were Comparatively, almost identical DLPE-derived (~4.1 µ m),GVs whichare the aresmallest slightly (~3.7 smaller than DMPCμm). When (~4.7 mixedµm). with Comparatively, DLPE, PCs/DLPE DLPE-derivedalso generate smaller GVs GVs, are ranging the smallest from 3.4 (~3.7 to µm). When3.8 mixed μm. As with for DLPE,the encapsulation PCs/DLPE efficiency, also generate although smaller internal GVs,calcein ranging can leak fromfrom inside 3.4 to 3.8 µm. As for the encapsulation efficiency, although internal calcein can leak from inside of the GVs to outside solutions, all GVs tested have ~90% or more vesicles encapsulated indicating that the w/o emulsion method is suitable for encapsulating materials inside of GVs. We then investigated the dynamics of the concentration (Figure3a) and size distribu- tion (Figure3b) of the above GVs (with or without calcein encapsulation) stored at 4 ◦C for Life 2021, 11, 223 9 of 13

indicated time periods up to one month. IFC analysis results showed that the yield of DLPE vesicles reduced almost by half at 6 h, and the concentration was the lowest at 3 d, to ~10%, indicating the instability of DLPE-derived GVs. The overall reduction of GV concentrations with DOPC or POPC fluctuated at a range of 20% to 40%. For GVs of DOPC:DLPE (7:3), the trend of the concentration changes at different time points is similar to DOPC vesicles. For GVs of POPC:DLPE (6:4), it is interesting to observe that the yields dropped to almost 20% at 12 h. Afterward, it increased to 1.8-fold at 1 w and then started to decline again (Figure3a). As shown in Figures3b and4, there was an apparent increase in the population of smaller size POPC:DLPE (6:4)-derived vesicles starting from 1 week, which could be the consequence of the long-term storage at 4 ◦C. For the whole size distribution of GVs, most GVs produced with DLPE, DOPC, POPC, and DOPC:DLPE were almost the same during Life 2021, 11, x FOR PEER REVIEW 12 of 16 the 1-month storage (Figure3b). Collectively, except for DLPE, GVs produced with DOPC, POPC, DOPC: DLPE (7:3), and POPC: DLPE (6:4) were stable.

Figure 3. StabilityFigure analysis 3. Stability of GVs. analysis The relative of GVs. concentrationThe relative concentration (a) and the (a size) and distribution the size distribution (b) of GVs(b) at different time points of GVs at different time points (0, 6 h, 12 h, 24 h, 3 d, 1 week, 2 ◦weeks, and 1 month) during (0, 6 h, 12 h, 24 h,storage 3 d, 1 week,at 4 °C. 2 weeks, and 1 month) during storage at 4 C.

To show a comparative analysis between IFC and microscopy, we further provide a series of observations under the confocal microscope, as shown in Figure 4. Basically, the fluorescence images could not give quantitive results, but rather trends for quality control. As experienced in our study, the results from microscopy images are greatly influenced by the resolution of the machine and the spot or area observed. For instance, fluorescent images showed that the yield of GVs of DOPC:DLPE (7:3) at 1 month seems higher than

Life 2021, 11, x FOR PEER REVIEW 13 of 16

at 0 h (Figure 4), probably due to the heterogeneity of GVs when we prepared the glass Life 2021, 11, 223 sample. Another example is that GVs of POPC:DLPE (6:4) became smaller at 2 weeks and10 of 13 1 month, but the concentrations showed almost no significant difference to the control (Figures 3a and 4).

FigureFigure 4. Confocal4. Confocal images images of highof high yield yield of GVsof GVs at different at different time time points points (0 h, (0 12 h, h, 12 24 h, h, 24 1 h, week, 1 week, and and 2 weeks) 2 weeks) during during storage storage at 4 ◦ C. at 4 °C. To show a comparative analysis between IFC and microscopy, we further provide a series4. Discussion of observations under the confocal microscope, as shown in Figure4. Basically, the fluorescenceRecently, images there couldhas been not a give lively quantitive interest in results, using butGVs rather to build trends either for protocells quality control. or Asartificial experienced cells that in ourmimic study, the biological the results functions from microscopy of cells [4,8,42]. images The are broad greatly application influenced byprospects the resolution of GVs ofincentivizes the machine the anddevelopmen the spott orof areaGV preparation observed. Formethods instance, and fluorescentstate-of- imagesthe-art showedanalytical that approaches the yield of[6]. GVs The ofw/ DOPC:DLPEo inverted emulsion-based (7:3) at 1 month process, seems compared higher than atwith 0 h (Figureothers such4), probably as gentle due hydration, to the heterogeneity electro-formation of GVs or microfluidic, when we prepared has benefits the glassof sample.high encapsulation Another example efficiency is that and GVs yield of of POPC:DLPE uni-lamellar (6:4)vesicles became with a smaller wide range at 2 weeks of lipid and 1compositions month, but the[15,27,28]. concentrations In this work, showed usingalmost the w/o no inverted significant emulsion-based difference method, to the control we (Figuresprepared3a GVs and4 with). different kinds of phosphatidylcholines (PCs) and phosphatidyleth- anolamines (PEs), which are two major of the plasma membrane. Once we 4.prepared Discussion the GVs, we firstly used a fluorescence microscope to observe them. Based on microscopy results, we can only detect whether or not the GVs formed and estimate the Recently, there has been a lively interest in using GVs to build either protocells or rough concentrations. artificial cells that mimic the biological functions of cells [4,8,42]. The broad application However, following the analysis of the imaging flow cytometer, which combines prospects of GVs incentivizes the development of GV preparation methods and state-of-the- high-resolution microscopy technology with traditional flow cytometry [15], we can per- art analytical approaches [6]. The w/o inverted emulsion-based process, compared with form extensive qualitative and quantitative imaging analysis of particles in a straightfor- othersward manner. such as gentleThe vesicle hydration, and non-vesicle electro-formation particles, orsuch microfluidic, as oil droplets has or benefits vesicle-oil of highmix- en- capsulationture, and vesicles efficiency with and or yieldwithout of uni-lamellarencapsulations, vesicles can be with divided a wide using, range via of imaging, lipid composi- all tionsvesicles [15, 27at, 28a single]. In this particle work, level. using Moreover, the w/o inverted vesicle emulsion-basedshape changes such method, as budding we prepared or GVs with different kinds of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), which are two major phospholipids of the plasma membrane. Once we prepared the GVs, we firstly used a fluorescence microscope to observe them. Based on microscopy results, we can only detect whether or not the GVs formed and estimate the rough concentrations. However, following the analysis of the imaging flow cytometer, which combines high- resolution microscopy technology with traditional flow cytometry [15], we can perform extensive qualitative and quantitative imaging analysis of particles in a straightforward Life 2021, 11, 223 11 of 13

manner. The vesicle and non-vesicle particles, such as oil droplets or vesicle-oil mixture, and vesicles with or without encapsulations, can be divided using, via imaging, all vesicles at a single particle level. Moreover, vesicle shape changes such as budding or bursting, vesicle fusion, or fission can also be detected and analyzed accordingly, depending on the experimental design. More importantly, the concentration, the size or size distribu- tion, and the encapsulation efficiency of GVs can be measured accurately, which increase reproducibility significantly. It has been reported in part that PC and PE, mainly egg PC [31,35–37], DOPC [33,36,37,43], POPC [17,44,45], DMPC [34,46], and DPPE [46] can be used to produce GVs by gentle hydration, electro-formation, or the w/o emulsion transfer method. By the gentle hydration method, DOPC/DOPE-PEG2000 [47], POPC/DPPE-PEG2000 [44] can form GVs; e.g., PC/POPE can prepare GVs by the electro-formation method [35]. So far, there is a lack of comprehensive study aiming at producing GVs with various kinds of PC and PE lipids. Here, we have prepared GVs with various kinds of single PC, PE, and PC:PE mixtures. We demonstrate that PCs including POPC, DOPC, DMPC, PEs containing DLPE, DMPE, and POPC:DLPE mixture, DOPC:DLPE mixture, and DMPC:DLPE mixture can yield a high production of GVs. The concentration and the size distribution of these GVs are relatively stable during lengthy time storage. Considering that cylindrical PCs and cone-shaped PEs promote vesicle-vesicle hemi-fusion [48], our findings will aid in vesicle preparation research, vesicle–vesicle fusion studies, and further developments of GVs in artificial membrane and artificial cell research.

Supplementary Materials: The following are available online at https://www.mdpi.com/2075-172 9/11/3/223/s1, Figure S1: The fluorescence microscope images of GVs prepared with all PCs and PEs used in this study, Figure S2: The fluorescence microscope images of GVs prepared with PCs: DLPE, Table S1: Characterization of vesicles produced with other lipids resulting lower yields. Author Contributions: Conceptualization, T.Y., and J.X.; methodology, B.X., and J.D.; Investigation, B.X., and J.D.; Writing—original draft preparation, B.X.; writing—review and editing, T.Y. and J.X.; validation, T.Y. and J.X. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Key R&D Program of China, Synthetic Biology Research, grant 2019YFA0904500; Chongqing Research Program of Basic Research and Frontier Technology, grant cstc2015jcyjBX0142 and cstc2016jcyjA0375; MOE International Joint Laboratory of Trustworthy Software at East China Normal University. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Acknowledgments: We appreciated Adriano Caliaria for proofreading and language editing of this manuscript. Conflicts of Interest: The authors declare no conflict of interests.

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