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Liposome-conta

BaBrUtiszcafa

Interdisciplinary Nanoscience Center (iNAN

E-mail: bstadler@inano.au.dk; Tel: +45 871

Cite this: Nanoscale, 2014, 6, 6426

Received 23rd January 2014Accepted 25th March 2014

DOI: 10.1039/c4nr00459k

www.rsc.org/nanoscale

6426 | Nanoscale, 2014, 6, 6426–6433

ining polymer films and colloidalassemblies towards biomedical applications

Boon M. Teo, Leticia Hosta-Rigau, Martin E. Lynge and Brigitte Stadler*

Liposomes are important components for biomedical applications. Their unique architecture and versatile

nature have made them useful carriers for the delivery of therapeutic cargo. The scope of this minireview is

to highlight recent developments of biomimetic liposome-based multicompartmentalized assemblies of

polymer thin films and colloidal carriers, and to outline a selection of recent applications of these

materials in bionanotechnology.

Introduction

In the last decade or two, liposomes have attracted signicantattention as potential drug carriers from researchers interestedin the eld of nanomedicine. These thermodynamically stablesupramolecular structures are formed spontaneously whenamphiphilic lipids are brought into contact with an aqueousphase. Once formed, liposomes are typically between 20 nm to10 mm in size with the phospholipid bilayer membrane beingapproximately 5 nm in thickness.1 The unique architecture ofliposomes allows for the loading of hydrophobic and hydro-philic therapeutic molecules; their charge and surface proper-ties can be tuned to enable stability during storage, control overthe drug-release rate, and to meet specic therapeutic needs.2

oon M. Teo is a postdoc fellowt iNANO, Aarhus University inrigitte Stadler's group. Sheeceived her PhD degree from theniversity of Melbourne, Aus-ralia in 2010. Her research liesn several complimentary areas,pecically, emulsion polymeri-ation, anisotropic Janus parti-les, proteinaceous microbubblegents and metal nanoclusterormation for theranosticpplications.

O), Aarhus University, Aarhus, Denmark.

5 6668

These nanocarriers are also biocompatible, since they are typi-cally made from lipids commonly found in biological systemsand biodegradable via the usual metabolic pathways. As such,the high biocompatibility and versatile nature of liposomeshave made these nanocolloids key components in manybiomedical research topics such as biomimetic chemistry, andhave been used as subunits in multilayered polymer thin lms(e.g. in ref. 3) and in colloidal assemblies for biomedicalapplications.4

In order to provide a succinct overview on the recent devel-opments in the use of liposomes as nanocontainers in thefabrication of subcompartmentalized functional layer-by-layer(LbL) surface coatings and multicompartmentalized colloidalassemblies (Scheme 1), this minireview is organized as follows.First we outline several subcompartments, namely, cyclodex-trins (CDs), block copolymer micelles (BCMs) and with focus onliposomes within polymer thin lms as examples of successful

Leticia Hosta-Rigau obtainedher PhD degree at the Universityof Barcelona in 2009 under thesupervision of Prof. F. Albericio.Her research was focused on thesynthesis of peptides with anti-tumor properties by solid phasepeptide synthesis. She thenstarted as a post-doctoralresearcher in the group of Prof.F. Caruso at the University ofMelbourne, Australia. Currentlyshe is a post-doctoral research

fellow at iNANO, Aarhus University, Denmark, where she receiveda Marie Curie individual fellowship. Her main interest is thedevelopment of cell mimics as a nature-inspired, functionalbiomedical platform with potential in enzyme replacementtherapy.

This journal is © The Royal Society of Chemistry 2014

Scheme 1 Schematic drawing of (a) polyelectrolyte multilayer filmscontaining liposomes and the interaction of cells with these polymer filmstowards SMDD, (bi) liposomes in liposomes (vesosomes) and (bii) cap-sosomes (capsule carrier containing liposomes as subcompartments).

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proof-of-concepts in surface mediated drug delivery (SMDD).Each of the subunits will be reviewed in terms of its assemblyand characterization towards SMDD. We will highlight thosecomposite coatings that are successfully used to deliver (active)compounds to adhering cells, and emphasize the few lmswhich have been tested in vivo and in vitro. Secondly, we willoutline and highlight several recent advances in colloidalassemblies containing liposomes as nanocontainers. We willdiscuss the assembly approaches employed to fabricate thesebiomimetic carriers and provide examples of successful proof-

Martin E. Lynge is currently aPhD student in Dr Stadler's lab.He received his MSc degree inmedicinal chemistry from Aar-hus University, Denmark in2012 for his work on liposome-containing polymer thinlms for surface-mediateddrug delivery applications.His current research projectconsiders the development ofsubcompartmentalized systemsthat can be utilised in either,surface-mediated or long circu-lating drug delivery.

This journal is © The Royal Society of Chemistry 2014

of-concepts of these colloidal assemblies as biomedicalplatforms.

Planar coatings

Planar lms consisting only of polymers are themselves able tocontrol cellular responses (e.g., cell adhesion and differentia-tion) or can be loaded with active cargo (e.g., proteins5 orgenes6,7) towards SMDD.8,9 However, trapping a small molecularpayload or cargo relying on its 3D structure such as enzymes,remains challenging. Trapping larger carriers such as drugdeposits to address this aspect has turned out to be a successfulapproach, especially when fundamentally different buildingblocks are employed, yielding subcompartmentalized multi-layered hybrid lms.

General overview

Cyclodextrins (CDs). CDs, cyclic oligosaccharides consistingof six (a), seven (b) or eight (g) glucose units within the ringedstructure results in a unique cup like morphology with a polarexterior and a hydrophobic interior,10 allow for the encapsula-tion of small hydrophobic molecules in an inclusion complexe.g., to enhance therapeutic effects or to reduce toxic side effectsof drugs.11–13 Therefore, modied CDs have been incorporatedinto multilayered lms,14,15 and these coatings can be engi-neered to be, for instance, responsive to solvent change,16 ordegradable by exposure to reducing conditions.17 There are alsoa limited number of reports where the biological evaluation ofthe CD-containing lms was considered, including the assemblyof pure anti-inammatory lms,18,19 anti-inammatory/micro-bicidal lms,20 lms with antitumoural activity,21 and lms forgrowth factors22 or gene delivery.23–25

Block copolymer micelles (BCMs). BCMs, assembled fromamphiphilic block copolymers, are powerful drug deposits withinpolymer lms which can either be glassy (i.e., hard and relativelybrittle)26–29 or responsive to environmental stimuli.30–35 There areonly single digit studies on BCM-containing lms whichdemonstrated drug delivery ability in vitro by delivering smallhydrophobic cytotoxic cargo,36 with so far no in vivo studies.

Brigitte Stadler obtained herPhD degree at ETH Zurich,Switzerland followed by postdoctime at the University of Mel-bourne, Australia. She has beenhead of the Laboratory for CellMimicry at iNANO, AarhusUniversity, Denmark since 2010.Her research group focuses onthe development and character-ization of smart, nature-inspiredassemblies towards their use intherapeutic cell mimicry as an

alternative approach to treat chronic medical conditions in asustained manner.

Nanoscale, 2014, 6, 6426–6433 | 6427

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Miscellaneous. In this section, examples of other types ofdrug deposits that have been trapped in polymer lms with theaim to be used in SMDD will be briey reviewed, although onlylittle biological evaluation has been reported. Cubosomes,cubic lipid mesophase nanoparticles,37 are one of the examplesas an alternative to liposomes.38 Polymersomes, assembledfrom amphiphilic synthetic block copolymers,39 have beenentrapped in planar polymer lms consisting of poly(vinylpyrrolidinone) and tannic acid by Lomas et al.,40 and in surface-adhering physical poly(vinyl alcohol) hydrogel lms by Hosta-Rigau et al.41 In the latter case, the viability of the cellsadhering to these coatings containing polymersomes loadedwith a hydrophobic cytotoxic drug was signicantly reduced ascompared to lms with empty polymersomes. Further, hydro-phobic nanodomains have been trapped in polymer lmswith demonstrated control of the release of encapsulatedpaclitaxel.42 In a recent study by the Lavalle group, vascularendothelial growth factor loaded degradable nanoparticleswere embedded within polyelectrolyte multilayers (PEMs).43

Enhanced proliferation and nitric oxide secretion has beendemonstrated for human umbilical cord vein cells adhering tolms containing the drug loaded particles.

Liposome-containing lms. Liposomes are by far the mostexplored drug deposits in polymer lms probably due to theirbiocompatibility, ease of preparation via self-assembly and theirinherent capability to entrap both hydrophilic cargo in theirlumen and/or hydrophobic cargo in their lipid membranes.

Assembly. The challenge of stably trapping intact liposomeswithout their displacement or rupture in polymer lms has beenaddressed in different ways. Katagiri et al. were the rst ones tocombine liposomes and polymer lms in 2002 by employingsilica-coated liposomes.44,45 Alternatively, intact liposomescoated with poly-D-Lysine were incorporated into polymerlms,46–49 including their use as microreactors.50 Furthermore,liposome trapping approaches without the need for stabilizationprior to their immobilization within the polymer coatingsemploy electrostatic interactions51,52 or non-covalent anchoringvia cholesterol-53–56 or oleic acid-55 modied polymers. In thelatter case, cholesterol turned out to be a more efficient anchorthan oleic acid or electrostatic interactions only to assemble(multiple layers of) liposomes in polymer lms.

Control over retention and release of cargo entrapped withinthe liposomes is of paramount importance for applications inSMDD. To this end, mechanisms to trigger the release of theircargo include temperature49,57 or electrochemistry.52

Drug delivery applications. To the best of our knowledge, wewere the rst ones in 2011 who employed liposome-containinglms in vitro towards SMDD by immobilizing zwitterionic lipo-somes (L and FL for uorescently labeled liposomes) onto a poly-L-lysine (PLL) pre-coated glass slide and subsequently cappingwith a poly(dopamine) (PDA) layer.60 PDA,3 deposited by oxida-tive self-polymerization of dopamine,61 is a versatile buildingblock in hybrid coatings with many unique properties includingthe possibility to control its thickness via the deposition condi-tions or the blending of DA with a (functional) polymer duringassembly.62,63 We demonstrated, that the cell mean uorescence

6428 | Nanoscale, 2014, 6, 6426–6433

(CMF) of adhering cells, due to their association with uorescentlipids trapped in the liposome membrane, could be controlledvia varying the thickness60 and type of capping layer.64 With theaim of facilitating the assembly, a mixture of liposomes anddopamine (F_10Mx: mixture of uorescent liposomes (FL) and xmg mL�1 dopamine) was successfully used to assemble lipo-some-containing lms with more sustained delivery of uores-cent cargo to adhering cells over 24 h depending on the assemblyconditions (e.g., dopamine concentration).59 Further, we deliv-ered the small hydrophobic and cytotoxic compound thiocora-line (TC) trapped in the lipid membrane of the liposomes fromthese lms to adhering myoblast cells and found a signicantdecrease in cell viability, demonstrating the potential of thesecoatings in SMDD.

Additionally, liposome-containing multilayered polymerlms were considered, and the response of adhering mamma-lian cells was assessed. Graf et al. used pH-sensitive, negativelycharged liposomes loaded with calcein trapped within polymerlayers, and demonstrated the uptake of the triggered releaseof the cargo by adhering myoblasts.65 We recently employedPLL/FL/PEMs (PEMs: (PLL/poly(methacrylic)-co-(cholesterylmethacrylate) (PMAc))x) or (poly(allylamine) (PAH)/poly(styrenesulfonate (PSS))x) lms, and evaluated the response of adheringmyoblasts and hepatocytes.58 The key ndings were that (i) theCMF of the hepatocytes, due to their association with uores-cent lipids, was higher than for myoblasts adhering to the samelm for the same time, (ii) the uorescence was evenly distrib-uted across the cell interior of the hepatocytes, while there wasan accumulation of uorescence in the myoblasts in the prox-imity of the nuclei (Fig. 1a), and (iii) the cell viability of adheringmyoblasts was found to be more reduced than for the hepato-cytes when TC was incorporated into the liposomes. In a followup, we combined the sequential deposition of polyelectrolyteswith PDA-based building blocks and assembled PLL/FL/PEMs/F_10M2.5 lms.59 An interesting nding was that there wasno signicant difference in the amount of adsorbed L depend-ing on the terminating polymer in the separation layers forPMAc or PSS, but the CMF of myoblasts adhering to lmsequipped with FL was signicantly lower in the latter case(Fig. 1b). This demonstrated that the access of the cells to theunderlying liposomes could be controlled via the lm assemblywithout the need for an active trigger.

In an alternative approach, we assembled liposome-con-taining surface adhering PVA hydrogels – lipogels.56 Wedemonstrated that cells could adhere to the lipogels when PLLor PDA was present and that the cells could associate withentrapped uorescent lipids. Further, paclitaxel as a smallhydrophobic cargo entrapped within the liposomes wassuccessfully delivered to the adhering cells from the lipogels,demonstrated by the reduced cell viability compared to cellsgrown on lipogels loaded with empty liposomes.

Recently, DeMuth et al. reported the assembly of polymer/liposome coated poly(L-glutamic acid) (PLGA) microneedles forvaccination purposes.66 In particular, liposomes loaded with anantigen, an adjuvant and a uorescent tracer for the lipid wallswere embedded within multilayers of biodegradable poly(b-amino ester) (PBAE) (Fig. 2a1). The microneedles were injected

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Fig. 1 Planar substrates: (a) representative confocal laser scanningmicroscopy images of myoblasts and hepatocytes adhering to PLL/FL/PMAc/(PEM)1 films for 24 h (FL: fluorescently labeled liposomes, PEM:(PLL/PMAc)3). The scale bar is 50 mm. Reprinted with permission fromref. 58. (c) CMF of myoblast cells adhering to PLL/FL/PMAc/(mSL)x/

F_10M2.5 films for 6 h and 24 h using different number of polymermultiple separation layers (mSLs) between the two liposome depositionsteps (F_10M2.5: 2.5 mg mL�1 dopamine mixed with FL solution).Reprinted with permission from ref. 59.

Fig. 2 (a) Schematic representation of PBAE/liposome multilayersdeposited onto PLGA microneedles. (1) Microneedles transfer (PBAE/ICL) coatings into the skin and hydrolytic degradation of PBAE leads toliposome release into the surrounding tissue (3), and delivery to skin-resident APCs provides coincident antigen exposure and immunosti-mulation for initiation of adaptive immunity. (b) SEM micrographs ofmultilayered-coated PLGA microneedles. Reproduced with permis-sion from ref. 66.

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into the cutaneous tissue of mice since it contains a highnumber of antigen presenting cells (APCs) (Fig. 2a2). The lipo-some containing lms were then transferred from the surface ofthe microneedles into the mice tissue and, upon degradation ofthe PBAE, the liposomes were taken up by the APCs, liberatingthe encapsulated antigens and adjuvants (Fig. 2a 3 and 4). Asignicant enhanced humoral immune response due to thetranscutaneous vaccination was observed. Fig. 2b shows a SEMmicrograph of the liposome coated microneedles.

Colloidal assemblies

Subcompartmentalized hybrid lms in colloidal form are rele-vant in therapeutic cell mimicry. This concept is inspired by thehierarchical structure of biological cells, which can performmultiple encapsulated reactions in a temporally and spatiallycontrolled manner in parallel or as a cascade.

General overview

The assembly of subcompartmentalized colloidal systems hasattracted considerable interest in the last few years. Conceptu-ally, single and multiple composition systems can be distin-guished, depending on whether the subunits and the carrier are

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assembled from the same or different type of components.The former case includes vesosomes,67 polymersomes withinpolymersomes,68–73 or polymer capsules within polymercapsules.74,75 The latter case includes polymer capsules con-taining cubosomes,38 polymersomes,40 liposomes (termed cap-sosomes) or polymer capsules together with liposomes.76

Although for many of these examples only the assembly hasbeen reported, it shows nonetheless the potential of compart-mentalized systems. However, within the scope of this minire-view, only concepts involving liposomes and subunits as drugdeposits will be further discussed. Therefore, approachesinvolving (metallic) nanoparticles e.g., for light triggered activityor imaging will not be considered and the reader is referred tothe recent review by Zasadzinski et al.77

Liposome-containing colloidal carriers

Assembly. Vesosomes,78 liposomes within liposomes, is asingle component system rst reported in 1997.79 The initialrather complex assembly approach has been replaced with asimplied protocol based on the trapping of small liposomesdue to the conversion of cochleate cylinders of dio-leoylphosphatidylserine sheets into micron-sized liposomes.80

Also, interdigitated lipid–ethanol sheets which close on them-selves when the temperature is increased, have been employedto entrap small liposomes.81 Vesosomes have demonstratedsuperior stability and control over cargo retention and release inbiologically relevant uids.82

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Fig. 3 Advanced capsosomes: schematic illustration (left) and fluo-rescent microscopy image (right) of microreactors containing lipo-somes and hydrogel capsules as subunits. Enzymatic conversion ratesof heterogeneous microreactors containing b-lactamase loadedliposomes in the presences (closed symbols) and absence (opensymbols) of Triton X. Reprinted with permission from ref. 76.

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Capsosomes are assembled in a similar manner as earlierdescribed for liposome-containing planar polymer lms but usingsacricial colloidal template particles. Cholesterol-based lipo-some anchoring to the polymer lm is again a core concept for theassembly. The polymer carrier capsules have predominantly beenassembled by using not only degradable PVP/thiolated PMA (e.g.,in Chong et al.83) but also non-degradable PAH/PSS.84 In theformer case, the type of the used crosslinker, to stabilize the thiolsto yield stable PMA hydrogel capsules under physiologicalconditions, depends on the encapsulating cargo. The oxidationagent chloramine T85 has proven to be a valid approach for cap-sosomes carrying hydrophobic cargo in the lipid membrane ofthe subunits,55,86 while more benign techniques were developedeither based on a thiol–disulde exchange,54,83,87 or by using smallcrosslinking molecules75,88–90 when enzymatic cargo was present.Stability and cargo retention are important properties for cap-sosomes. In the former case, PEGylation of the outer membraneof the carrier capsules not only improved their fouling charac-teristics, but also reduced the enzymatic degradation of the lipo-somal subcompartments by phospholipase A2.90 In the latter case,the lower limit of the size of cargo for stable encapsulation wasfound to be between 400 and 550 Da,91 with good long-termstability for larger molecules like enzymes.53,87

Activity. This paragraph reviews those assemblies, whichhave demonstrated functionality relevant for biomedicalapplications i.e., for intracellular drug delivery or for encapsu-lated catalysis towards therapeutic cell mimicry.

Intracellular drug delivery. Admittedly, the assemblies dis-cussed here are rather large for intracellular drug delivery, andtheir use for intravenous administration is rather limited.Nonetheless, their interaction with cells/tissue is an interestingaspect to consider in the right settings.

Fusogenic vesosomes were considered for transcutaneousimmunization and evaluated in cell culture and in vivo.92 Theantigen tetanus toxoid (TTx) was encapsulated in the liposomalcompartments. They found a signicantly increased level ofanti-TTx antibodies in vivo when TTx was topically delivered viathe fusogenic vesosome system as compared to cationic uni-lamellar liposomes or the free antigen. This study demon-strated the potential of liposome based single componentsystems in drug delivery.

Although intracellular drug delivery was never the coreapplication considered for capsosomes, assessing the interac-tion of capsosomes with the cell becomes valid when consid-ering other administration pathways than intravenous injection.The ability to encapsulate small lipophilic cargo in the subunitswas conrmed using TC86 and taxol (TX, a mitotic inhibitor),55

and the amount of encapsulated cargo was conrmed to bedependent on the number of liposome deposition steps. Thereduction in viability of cells upon exposure to capsosomesloaded with the cytotoxic compounds TC or TX was conrmed,while pristine capsosomes did not exhibit intrinsic toxicity,making them promising candidates for biomedical applications.

Encapsulated catalysis. The performance of encapsulatedcatalysis in subcompartmentalized systems is a promising way tomimic (simple) metabolic activities. Among the rst ones toexplore this concept using liposomes was the Vogel group.

6430 | Nanoscale, 2014, 6, 6426–6433

Bolinger et al. employed surface-tethered large unilamellar lipo-somes with entrapped small unilamellar liposomes to demon-strate consecutive enzymatic reactions in a single nanoreactor.93

Encapsulated enzymatic reactions within capsosomes wereused with the aim to mimic metabolic activities. Initially, theenzyme b-lactamase was employed to convert the substratenitrocen into its hydrolyzed product, using the surfactantTriton X to lyse the liposomes and to start the reaction.94 Infollow up work, we conrmed that the reaction kinetics corre-lates with the amount of encapsulated enzymes, and thattemperature can be used to trigger the reaction allowing for therecycling of the capsosomes.89,91 However, most biologicallyrelevant reactions are more than single-step conversions butmost oen involve multi-step cascade reactions runningsimultaneously within the same carrier. To date, one of themost advanced synthetic assemblies relies on the immobiliza-tion of different enzymes at different positions of the carrier i.e.,in the void, inside the membrane, and on the surface.95,96 Withthe aim to further push the complexity of capsosomes, triggeredpeptide cargo release using encapsulated enzymatic catalysiswas demonstrated, by enzymatically converting glutathionedisulde into its reduced form, glutathione which was able to

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cleave the disulde bond that linked the peptide to a polymercarrier.88 The assembly of a heterogeneous microreactor con-taining liposomes and hydrogel capsules as subunits has beenreported (Fig. 3, top).76 The functionality of these heterogeneousmicroreactors has been demonstrated by controlled degrada-tion of the polymeric subunits and the preservation of theactivity for liposome encapsulated b-lactamase (Fig. 3, bottom).

Conclusions/future perspective

In this minireview, we summarized and highlighted the recentprogress of employing drug deposits, namely, CDs, micelles andliposomes, assembled on 2D planar surfaces and 3D colloidalcarriers. Focus was put on liposomes since this type of drugdeposit is particularly interesting due to its distinguishedadvantages over CDs and micelles, including the facts thatliposomes can be carriers for hydrophilic and hydrophobiccargo, a diverse range of lipids is commercially available or theiruse in drug delivery has accumulated broad fundamental andapplied knowledge. Although there is enormous potential forthe translation of these substrates containing nanocontainerstowards biomedical applications, the number of in vitro studiesreported is scarce with even rarer in vivo studies being reported.Further, long-term effects and sustained delivery has also yet tobe realized for many systems. The eld is still in its infancy butthe potential is tremendous, reected by the growing number ofscientic reports each year. The focus will have to shi fromintroducing different assembly concepts to implementingfunctions into the lms such as controlled loading of drugdeposits and desirable sustained cargo release to adhering cells,imposing a positive therapeutic effect. We expect that in thenext few years, there will be rapid progress and development inthis eld particularly in performing in vitro and in vivo studiesand their wide implications in medical therapies. Hence, wehope that this minireview will inspire multidisciplinaryresearch interest in this fascinating topic and to bring thesesubstrates a step closer to clinical trials.

Acknowledgements

This work was supported by a Sapere Aude Starting Grant fromthe Danish Council for Independent Research, Technology andProduction Sciences, Denmark and a Marie Curie individualfellowship.

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