Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell

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Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models Nan-Nan Deng, Maaruthy Yelleswarapu, Lifei Zheng, and Wilhelm T. S. Huck J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b10977 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 17, 2016

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Journal of the American Chemical Society

M Microflu uidic Asse embly of o Monod disperse Vesosomes as A Artificial Cell M Models N Nan-Nan Deeng, Maaruth hy Yelleswarrapu, Lifei Zheng Z and W Wilhelm T. S S. Huck* R Radboud Univeersity, Institutee for Moleculess and Materialss, Heyendaalseeweg 135, 65255 AJ Nijmegenn, The Netherlaands

SSupporting Info formation A ABSTRACT: Vesosomes V are nested liposomaal structures wiith hhigh potential ass advanced drug delivery vehiclees, bioreactors an nd aartificial cells. However, H to datee no method hass been reported to pprepare monodisperse vesosom mes of controlleed size. Here we w rreport on a mu ulti-step microflluidic strategy for hierarchicallly aassembling uniform vesosomees from deweetting of doub ble eemulsion templates. The contrrol afforded by y our method is illustrated by the t formation of concentric, pericentric an nd m multicompartmen nt liposomes. The microfl fluidic route to vvesosomes offerrs an exceptionaal platform to bu uild artificial cells aas exemplified by the in vitro traanscription in “nu ucleus” liposom mes aand the mimicry y of the architeccture of eukaryo otic cells. Finallly, w we show the trransport of smaall molecules across a the nucleeic eenvelope via inseertion of nanopo ores into the bilay yers.

Vesosomes are multicomparrtmental liposom mal structures of vvaried architectu ures.1 These mulltivesicular vesiccles can consist of m multilayered co oncentric lipossomes, or mu ultiple liposom mes aarranged within or around largee liposomes. Th hese structures, as w well as relatted multicomp partment poly ymersomes2 an nd 3 pproteinosomes, have generateed significant interest i for theeir ppotential as drug g delivery vehiclees1a,4, compartm mentalized nano- or m microscale bioreeactors,5 or as artificial cell/p protocell models.6 Z Zasadzinski and d coworkers haave shown that vesosomes can pprotect encapsulated drug carrrying compartm ments from bloo od ccomponents due to the presence of double bilayeers,4b and slow th he rrelease of vesiicle contents.4cc Bolinger et al.5b,c employed m multicompartmen nt vesosomes to o perform conseecutive enzymattic rreactions by theermally remote release of diffferent compoun nds eencapsulated in smaller liposom mes. Remarkably y, vesosomes th hat ccontain a “nuclleus” or “organ nelles” represen nt a new conceept ttowards the design of artifi ficial cells, esp pecially artificiial eeukaryotic cells.6a However, con nventional metho ods,7 such as bu ulk hhydration8 or eleectroformation9 of dried lipid membranes, m do not n rreadily yield complex c hierarcchical vesicularr structures, an nd ccertainly do not provide any co ontrol over dimeensions, offer lo ow eencapsulation efficiencies e and d low yields. Moreover, theese sstrategies do no ot allow a systtematic simultan neous loading of ddifferent comp ponents into diverse comp partments. Theese limitations have severely hinderred progress in the application of ssuch multivesicu ular liposomes. Recently, drop plet microfluidiics hhas been shown n to offer excelllent emulsion templates for th he ppreparation off monodispersse liposomes100 as well as ppolymersomes2,11, but no vesosomes have been reported. r Recently, we have presented a surfactant-asssisted microfluid dic sstrategy for asseembling monodiisperse liposomes from water-iin-

oil-in-w water (W/O/W) double emulsion droplets.10a Inn this paper, we dem monstrate the hierarchical assem mbly of uniform m vesosomes via muulti-dewetting pprocesses. Briefly, the singlee liposomes formedd from dewettinng of microfluuidically preparred W/O/W emulsioon droplets weree reinjected into microfluidic deevices as the inner pphase to fabricatee larger liposom me-loaded W/O/W W emulsion dropletts, which will uundergo a secoond dewetting trransition to form unniform liposomee-in-liposome veesicles. The conttrol afforded by ourr method is illuustrated by thee formation of concentric, pericenntric and multiccompartment lipposomes. Finallyy, we show the resuultant vesosomees are potentiallyy used as compllex artificial cell moodels.

Figure 1. (a,b) Schem matics and snappshots of the m microfluidic preparaation of double emulsions with an inner liposoome and the assembbly of vesosomees from emulsionn dewetting. (c,dd) Confocal images of the monodissperse vesosomees with one, twoo, three and four innner liposomes. ((e) The size disttribution of inneer and outer liposom mes of the vesossomes in c. (f) A As-formed vesoosomes with differennt-sized interior liposomes. Scalle bars, 100 µm. To pproduce monoddisperse vesosoomes, we firstt fabricated monodiisperse single liiposomes as thee “nuclei” accorrding to our recent publication (Figure S1).10a Suubsequently, thee preformed single liposomes disppersed in an aqqueous phase (W W1, see SI were reinjected iinto the microfluuidic device Materiaals for details) w

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aas inner phase to make larger W/O/W W double emulsion dropleets ((Figures 1a, b1, S2 and Moviess S1 and S2, seee SI Materials for f ddetails of the middle m oil phasee (O) and the outer o water phaase ((W2)). In this way, w smaller lipo osomes were su uccessfully loaded into the inner droplets d of dou uble emulsions. As we reported ppreviously,10a th he dewetting transition is induced by th he sspontaneous redu uction of the interfacial energiess of the W1/O/W W2 eemulsion system m, i.e., a positiv ve spreading coeefficient of phaase W W2. Along with h the evaporation n of chloroform m in the oil phasse, tthe oil shells off the larger dou uble emulsion droplets d graduallly ddewet from the interior drops (Figures 1b2 and a S3), formin ng vvesosomes and separated oil drops containiing excess lipiids ((Figures 1b3 and a S3). To visualize the veesosomes clearlly, ffluorescein issothiocyanate-deextran (FITC C-Dextran) an nd rrhodamine B isothiocyanate--Dextran (RITC-dextran) weere rrespectively used d to label the in nner and outer liiposomes (Figurres 11c,d,f and S3). As A Figure 1c sh hows, the resultaant vesosomes are a uuniform; their internal and extern nal mean diametters are 43 µm an nd 1102 µm respectiv vely and their co oefficients of varriation are 5% an nd 33% respectively (Figure 1e). Nottably, the yield of o vesosomes wiith ssingle inner lipossomes is as high h as 66.4% (Figu ure S5). This microflu uidic method gives control ov ver the vesosom me ddimensions and d configurationss. The numberr of the interiior liposomes can bee varied by tunin ng the flow ratess. For example, we w ffixed the flow raates of middle an nd outer phase att 300 and 2500 µL µ h-1, and adjusted d the inner phasee flow rates from m 150, 280 to 36 60 µ µL h-1 to preparre vesosomes thaat mainly contaiin one and two to ffour inner liposo omes (Figures 1cc,d). The distrib bution of numbeers oof encapsulated d liposomes sh hows an overalll growing tren nd ((Figures S4-S9),, and the yields of each main products p are mo ore tthan 20%. Besid des, by slowing down d the flow raate in outer phasse, w we also preparred vesosomes that encapsulaate 3 to 5 inn ner liposomes (Figu ures S10 and S1 11). In addition n, vesosomes wiith ddiverse dimensio ons of sub-comp partments were also a fabricated. As A F Figures 1f and S12 S show, the sh hell volumes bettween interior an nd eexterior liposom mes can be adjusted, which allow w the precise load oof ingredients. ofluidic techniique has show wn Further, the droplet micro cconsiderable flexibility in encaapsulation of diistinct droplets in m multiple emulsio on droplets,12 which w offers perrfect templates to ccreate structured vesosomes. To demonstratte this idea, we w uupgraded the microfluidic m device with two in ndependent inleets ((Figure 2a),10a which w were emplloyed to preparee double emulsio on ddroplets with different d liposomes. Similarly,, these emulsio on ttemplates underg go a complete dewetting d to geenerate vesosom mes ccontaining distin nct liposomes. As A Figure 2b shows, s vesosom mes w with diverse inn ner structures were w successfully y achieved. Mo ore tthan 300 vesoso omes were colleected and analyzzed, showing th hat 332% and 22% of the vesosomess contain 2 and 3 inner liposom mes rrespectively; thee rest are empty y, or contain on ne or more than n 4 inner liposomes (Figures 2c and d S13). We note that the pairing of rreinjected liposo omes is a key point p to generatee vesosomes wiith ccontrolled numbers and types off inner liposomess. The method can bbe extended to make m more comp plex concentric multicompartme m ent vvesosomes, forr example, liposome-in-lipo l osome-in-liposom me vvesosomes (Figu ures 2 d and e). The vesosomees essentially rep present liposomees which contain na ““nucleus” or “organelles”, and reepresent a prom mising new conceept ttowards buildin ng artificial cells, especiallly the artificiial eeukaryotic cells. Although liposo omes have been n widely employed aas artificial cellls to imitate the architectures and functions of pprokaryotic cellss, such as cell div vision,13 RNA reeplication14 and in vvitro transcripttion/translation (IVTT),6c th he mimicry of intracellular co ompartmentalizaation both in structures an nd ffunctions remain ns underexplored, mostly becau use the high-ord der vvesicles required d, are difficult to make. Furtherm more, convention nal

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routes tto the vesosome s typically do noot efficiently co--encapsulate differennt solutions intto different subb-compartments. Here, we show tthe advantages of our microflluidic approach in loading diversee complex compponents and thee potential of thhe resultant vesosom mes in building artificial cells.

Figure 2. (a) Schematiics of the microffluidic preparatioon of double emulsioons with distinnct interior lipoosomes and thee dewetting processs. (b) Confocal images of thee vesosomes wiith different numberrs and ratios of iinterior liposomees. Inset in b is aan emulsion templatte undergoing deewetting. (c) Diistribution of thee number of inner liiposomes of as--formed vesosom mes (Figure S133, N= 329). (d,e) T The formation of triple vesoosomes and thhe resultant structurres. Scale bars, 1100 µm.

Figure 3. (a,b) Cartoonns showing in siitu detection of IIVTx inside NA aptamer the lipoosomes (a) and tthe working prinnciple of the RN Spinachh2 (b). (c) Sequuence of images showing syntheesis of RNA in the liposomes. (d) The RNA expression kinetics. (e) Indepenndent encapsulaation of IVTx m mix into interiorr liposomes and IV VTT mix into exterior liposom mes. (f) IVTx in nucleus liposom me of vesosomess. Scale bars, 50 µm.


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F Figure 4. (a) Caartoons and (b) confocal c image series s show proteein pore-mediateed transport of ffluorescent moleecules from inneer liposomes tto outer liposomes. (c,d) Controll experiment: no o melittin was ad dded. (e) Kineticcs of time-depenndent release of ccalcein from innner liposome tto outer liposomee. The curves 1, 2, 3 and 4 refer to the fluorescen nce in relevant lliposomes as shoown in figures a and c. Scale barrs, 50 µm. To mimic the key function of cell nuclei, we encapsulated e an in vvitro transcriptio on (IVTx) mix in i single liposom mes to synthesize R RNA (Figure 3a)). This mix conttains T7 RNAP, pyrophosphatasse, D DNA template, RNase R inhibitorr and feeding bu uffer (see SI IVT Tx in nucleus lipossomes & IVTT for details), an nd all componen nts w were successfully loaded in in nner droplets during d the doub ble eemulsion templaate preparation. To T visualize thee RNA generatio on, w we coded the plaasmids with a seequence for Spin nach2 aptamer th hat ccan bind a dye called 5-difluoro-4-hy ydroxybenzyliden ne imidazolinone (DFHBI) ( to fo orm a fluoresccent complex of S Spinach2-DFHB BI (Figure 3b); both DFHBI and a Spinach2 are a nnon-fluorescent until u binding occcurs.15 As Figurres 3c, d, S14 an nd M Movie S4 show, the fluorescence intensity of Sp pinach2-DFHBI in liposomes notably increased from m 0 min to 60 min m and up to 12 20 m min during the IVTx. After 2 h expression, the fluorescence ssignal reached a plateau probablly due to runnin ng out of nutrien nts ((Figure 3d). Th his agrees well with the IVTx reaction in bu ulk rrecorded by a plaate reader (Figurre S15). To mimic the architecture of eukaryotic e cells, we embedded th he ssingle liposomess that contain IV VTx mix as arttificial nuclei in nto larger liposomess. In the larger droplets, d IVTT mix m (consisting of oone-third Escheerichia coli cell lysate and tw wo-thirds feedin ng bbuffer, see SI IV VTx in nucleus liposomes & IV VTT for details))16 ffor monomeric red r fluorescent protein p (mRFP) was encapsulateed tto form a kind of “cytoplasm m” phase (Figure 3e). RNA was w ssuccessfully exp pressed inside th he nucleus liposo omes as shown in F Figures 3e (last image) and 3f. Moreover, mosst of the artificiial ccell models are stable for more than 8 h, which is sufficient for f IIVTT or related research. Recen ntly we already demonstrated th he iin vitro expresssion of enhanced green fluoreescent protein in liposomes.10a Ass we here show that transcriptio on and translatio on ccan in principle be carried out in i two differentt compartments of tthe artificial cellls, the final key (and probably most m challengin ng) sstep would be to realize the transsfer of RNA acro oss lipid bilayerss. d passage of nu utrient molecules and wastes iss a The controlled kkey feature of biological b cells, in which the nanopores n play an a important role in n both cell envellops and nucleic shells. Thereforre, tto demonstrate the t feasibility of molecule transsfer between su ubccompartments, we w inserted a membrane protein n melittin into th he nnucleus liposom mes to create naanopores. Melitttin self-assemblles into bilayers to create c pores of 1-3 1 nm or 3.5-4.5 nm in diametter

dependding on the num mber of assemblly subunits,17 w which allows small m molecules to traansfer. As Figurre 4a shows, wee loaded the inner lliposomes with melittin monoomers (2 μM) aand calcein fluoresccent molecules (Mw= 623 g mol-1, 10 μM)) and outer liposom mes with RITC-D Dextran (averagge Mw= 70000 g mol-1), and then obbserved the fluuorescence intennsity over timee. Once the nanopoores were formeed in the nucleeus bilayers, thee green dye (calceinn) in the inner liposomes rapiddly (about 2 miin) diffused into thee outer liposomees, while the redd dye (RITC-Dexxtran) in the outer liiposome failed to diffuse into the inside because of the larger m molecule weightt (Figure 4b, Moovie S5). This sizze exclusion phenom menon also indiicates that the morphologies oof both the inner aand outer liposom mes are intact, eensuring that thhe release of the fluuorescent molecuules is not because of the burrst of inner liposom mes. After the ddye diffusion, thhe distribution oof calcein in the vessosomes is hom mogeneous (Figuure 4b calcein cchannel and Figure 4e curves 1 and 2). In coontrast, in the absence of was observed eveen after 2 h membrrane proteins, noo dye transfer w (Figurees 4c-e, Movie S S3). Furthermorre, it has been reeported that insertioon of other meembrane proteinns into the bilaayers forms nanopoores of different sizes, for exampple, alpha-hemolysin (αHL) creates pores of 1.4 nm m in diameter.188 Therefore, it iss feasible to selectivvely insert diffferent membranne proteins, foor example, melittinn and αHL, into internal and external liposomes respecttively to createe different nanoopores, which w will endow differennt vesosome c ompartments w with different ppermeability propertties. This willl open up platforms to study the commuunications betweeen artificial ceell compartmennts and that betweeen compartment and surroundingg environment. microfluidic In ssummary, we hhave reported a multi-step m strategyy for the hierarcchical assembly oof uniform vesoosomes from dewettiing of W/O/W double emulsiion droplets. Too show the controll over vesosom me formation, cconcentric, pericentric and multicoompartment lipoosomes were suuccessfully creaated. These structurres offer an addvanced platforrm to build arrtificial cell modelss as exemplifieed by the encaapsulation of ssolutions as compleex as that forr IVTx and IVTT into thhe different comparrtments, and reeal time detecction of RNA generation. Finallyy, we showed thhe insertion of nnanopores into the nucleus mes, which alloows for the trannsport of smalll molecules liposom across the envelopes.. Future work will focus onn nanoporeRNA across bilaayers and coupled IVTT in mediateed transfer of R



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vesosome systems. We believe that this approach for fabricating uniform vesosomes with well-defined sizes, will facilitate artificial cell related research like enzyme storage and release, membrane fusion, vesicle transport and multi-step bioprocesses.

ASSOCIATED CONTENT Supporting Information Supplementary experimental section, figures and movies are available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION Corresponding Author *Wilhelm T.S. Huck ([email protected])

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the Netherlands Organization for Scientific Research (NWO, TOPPUNT grant 718.014.001, the Ministry of Education, Culture and Science (Gravity programme, 024.001.035), and a Marie Skłodowska-Curie Actions Individual Fellowship to N.-N.D. (EC, H2020-MSCA-IF grant 659907). We thank Sandra Wardle (Radboud University) and Laure Eydieux (Polytech Clermont-Ferrand) for their help with the RNA aptamer preparation.

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Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell

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