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Cholesterol – a biological compound as a building block in bionanotechnology

Rigau, Leticia Hosta; Zhang, Yan; Teo, Boon M.; Postma, Almar; Städler, Brigitte

Published in:Nanoscale

Link to article, DOI:10.1039/c2nr32923a

Publication date:2013

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Rigau, L. H., Zhang, Y., Teo, B. M., Postma, A., & Städler, B. (2013). Cholesterol – a biological compound as abuilding block in bionanotechnology. Nanoscale, 5, 89-109. https://doi.org/10.1039/c2nr32923a

https://doi.org/10.1039/c2nr32923a

https://orbit.dtu.dk/en/publications/cholesterol--a-biological-compound-as-a-building-block-in-bionanotechnology(e46bfbd4-e223-46e5-a5cc-1e279d400b56).html

https://doi.org/10.1039/c2nr32923a

Nanoscale

FEATURE ARTICLE

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Cholesterol – a bio

LPUiorstP2doD

Biomolecular Engineering at thetralia. Currently she is a post-doctAarhus University, Denmark, wheindividual fellowship. Her mainarticial cells towards a functiopotential in enzyme-therapy.

aiNANO, Aarhus University, Aarhus, Denmar

8715 6668bState Key Laboratory for Modication of

College of Material Science and Engineering

People’s Republic of ChinacCSIRO Materials Science and Engineerin

Clayton, Victoria 3168, Australia

Cite this: Nanoscale, 2013, 5, 89

Received 26th September 2012Accepted 31st October 2012

DOI: 10.1039/c2nr32923a

www.rsc.org/nanoscale

This journal is ª The Royal Society of

logical compound as a building blockin bionanotechnology

Leticia Hosta-Rigau,a Yan Zhang,ab Boon M. Teo,a Almar Postmac

and Brigitte Stadler*a

Cholesterol is a molecule with many tasks in nature but also a long history in science. This feature article

highlights the contribution of this small compound to bionanotechnology. We discuss relevant chemical

aspects in this context followed by an overview of its self-assembly capabilities both as a free molecule

and when conjugated to a polymer. Further, cholesterol in the context of liposomes is reviewed and its

impact ranging from biosensing to drug delivery is outlined. Cholesterol is and will be an indispensable

player in bionanotechnology, contributing to the progress of this potent field of research.

Introduction

Bionanotechnology is aiming to learn from nature’s approachto assemble and run “nano-machines” and to mimic theseconcepts by engineering functional nano-devices e.g. makinguse of the self-assembly capability of biological building blocksor imitate nature’s way of motility.1–3 In the latter case, attempts

eticia Hosta-Rigau received herhD in Chemistry from theniversity of Barcelona, Spain,n 2009, under the supervisionf Prof. F. Albericio. Heresearch was focused on theynthesis of peptides with anti-umor properties by Solid Phaseeptide Synthesis. During 2010–011 she worked as a post-octoral researcher in the groupf Prof. F. Caruso at theepartment of Chemical andUniversity of Melbourne, Aus-oral research fellow at iNANO,re she received a Marie Curieinterest is the development ofnal biomedical platform with

k. E-mail: [email protected]; Tel: +45

Chemical Fibers and Polymer Materials,

, Donghua University, Shanghai 201620,

g, Ian Wark Laboratory, Bayview Ave,

Chemistry 2013

to copy the agella of bacteria4,5 or the use of motor proteins6

have been considered. In the former case, the self-assembly ofDNA,7,8 peptides,9,10 or lipids11,12 into a variety of nanostructureshas been demonstrated and their complexity is permanentlyincreasing and functionality is implemented. Conjugates withbiomolecules e.g. cholesterol, have proven to be indispensablebuilding blocks. Cholesterol, an essential component ofmammalian cells, has been described by Michael S. Brown andJoseph L. Goldstein as “the most highly decorated small mole-cule in biology”,13 and this statement could probably beextended for its application in bionanotechnology as well.

Looking back, the term “cholesterol” was introduced in thebeginning of the 20th century from the term “cholesterine”,coined by Chevreul in 1816 “Je nommerai cholesterine, de colh,

Yan Zhang is currently a PhDstudent in the College of Mate-rial Science and Engineering atDonghua University, China.Prior to this, she received her MSdegree in Tianjin PolytechnicUniversity, China. Currently,she works as an exchange PhDstudent in the group of AssistantProf. Brigitte Stadler at iNANO,Aarhus University, Denmark.Her research involves theassembly of advanced capsulestowards their application indrug delivery.

Nanoscale, 2013, 5, 89–109 | 89

http://dx.doi.org/10.1039/c2nr32923a

http://pubs.rsc.org/en/journals/journal/NR

http://pubs.rsc.org/en/journals/journal/NR?issueid=NR005001

Nanoscale Feature Article

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bile, et 23r3o2, solide, la substance cristallisee des calculs biliaireshumains” (I will call cholesterin of colh, bile, and 23r3o2, solid,crystalline substance of human gallstones).14 However, choles-terol as a substance was already known in the late 1700’s as analcohol soluble fraction separated from human gallstonesobserved by Poulletier de la Salle. In 1789 De Fourcroy hadprepared a large quantity of this substance, believing that it isthe same as “blanc de baleine” or spermaceti.15 He labeled thecrystalline fraction “adipocire” or fatty wax, a material similar tothat obtained from acid treated grave wax. (Grave was asubstance formed normally in damp areas of cemeteries fromthe decomposition of cadavers.) Chevreul later showed that itdiffered from these compounds by treatment with potassiumhydroxide and melting point comparisons.16

Initial forays into elucidation of the structure of cholesterolshowed it to be an alcohol17 in the course of the preparation ofesters, and later it was shown to be a secondary alcohol18

through its conversion into the ketone cholesterone. The pres-ence of the double bond was established in 1868. Furtherstructural elucidation through parallel studies on derivatives ofcholesterol and chemically related bile acids, with verication

Boon M. Teo received her PhDfrom The University of Mel-bourne, Australia in 2010working on ultrasonic polymersynthesis. Currently, she isworking as a post-doc in AssistantProf. Brigitte Stadler’s group atAarhus University, Denmark. Herresearch interests include emul-sion polymerization, anisotropicpolymer colloids and drugdelivery systems.

Almar Postma received his PhDfrom The University of NewSouth Wales, under the supervi-sion of Prof. T. P. Davis and DrsG. Moad & M. O’Shea (2005) inthe elds of controlled radicalpolymerisation and reactiveextrusion. From 2005 to 2008 heheld a postdoctoral fellowshipposition with Prof. F. Caruso atThe University of Melbourneworking on both polymer-basedcapsular drug delivery vehicles

and with a startup company (iCeutica) in pharmaceutical nano-particulate reformulations. He re-joined CSIRO as a ResearchScientist in 2008. His current research interests lie at the interfaceof polymer design/synthesis and their applications in nano-biomedicine and electoactive materials.

90 | Nanoscale, 2013, 5, 89–109

by X-ray studies,19 allowed the established formula to be arrivedat as described in reviews by Windaus and Neukirchen.20,21

In nature, cholesterol is heterogeneously distributed incellular membranes, with the highest content found in theplasma membrane.22 Most nucleated cells synthesise choles-terol in a regulated process consisting of multiple steps thatmainly take place within the endoplasmic reticulum (ER).23–25

Alternatively, cholesterol can be taken up by the cells fromlipoproteins.26 There are various pathways to traffic cholesterolto the plasma membrane in order to maintain the highconcentration.27 When the cholesterol level reaches a thresholdlevel which is regulated by the sphingomyelin content of thecell, cholesterol is transported to the ER for esterication andstorage.28

The rst link between cholesterol and human health wasdiscovered in 1843, when Vogel showed that cholesterol was amajor component of arterial plaques,29 and it has since thenbeen intensively investigated.30,31 Cholesterol was consequentlydetermined to be widely distributed in the animal kingdom andits isomeric forms, phytosterol, were shown to frequent thevegetable kingdom.32 Sterol research in the mid- to late 20th

century progressed to biomimetic chemistry,33 tracer work,34

advanced structural determination of cholesterol using high-eld NMR and X-ray diffraction methods,35 with a focus on theregulation of cholesterol synthesis and human physiology.36,37

Cholesterol’s predominant characteristic is its ability tomodulate the physicochemical properties (e.g. uidity23 andpermeability38) of the cell membrane. Due to this, cholesterolaffects a number of cellular processes. For instance, cholesterolinuences the behaviour of membrane proteins e.g. too muchcholesterol and ordering will slow down the diffusion ofmembrane proteins and increase the bending modulus of themembrane.39 Cholesterol also acts as a precursor for thesynthesis of bile acids40 (which are stored in the gallbladder andhelp to solubilise fats), and is also important for the metabo-lism of fat soluble vitamins, including vitamins A, D, E, and K.41

Brigitte Stadler received her PhDfrom ETH Zurich, Switzerland,followed by over two years as apost-doc in the group of Prof. F.Caruso at the University ofMelbourne, Australia. Since2010, she is Assistant Professorat iNANO, Aarhus University,Denmark aer she obtained aSapere Aude Starting Grant fromthe Danish Council for Inde-pendent Research, Technologyand Production Sciences. Her

research group works on the development and characterization ofsmart nature-inspired drug delivery vehicles and on the estab-lishment of microuidic platforms to address questions inbiomedicine.

This journal is ª The Royal Society of Chemistry 2013


Feature Article Nanoscale

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It is the major precursor for the synthesis of vitamin D andvarious steroid hormones.42 Recently, cholesterol has also beenfound to play an important role in membrane trafficking, sort-ing processes, and cell signalling processes, where researchershave suggested that it is involved in the formation of lipid rasin the plasma membrane, as represented by G protein-coupledreceptor signalling.39,43,44

The aim of this feature article is to outline the impact thatthe small biomolecule cholesterol has made in the eld ofbionanotechnology (Scheme 1). We are reviewing aspects andprogresses in this area made possible and/or considerablyimproved and facilitated due to the presence of cholesterol. Inthe rst part, we will outline the chemistry of cholesterol,starting with fundamental aspects, followed by cholesterolmodications and, nally, the synthesis of cholesterol-con-taining polymers will be discussed. The next section willaddress the state-of-the-art and challenges involving the self-assembly of cholesterol and cholesterol-modied polymers intonanostructures. The last part of this feature article will considerthe effect of cholesterol on liposomes in bionanotechnologyeither as a stabiliser in drug delivery and in analytical science,or as an anchoring unit in engineering of biosensors, for thedecoration of liposomes with polyethylene glycol (PEG), or inthe assembly of capsosomes. The involvement of cholesterol ineach of these aspects will be demonstrated by giving selectedexamples. Overall, we hope to demonstrate that the smallmolecule cholesterol contributes signicantly to the eld ofbionanotechnology and its many applications.

The chemistry of cholesterol

Cholesterol is an amphiphatic molecule, a compound pos-sessing both hydrophilic and lipophilic properties. It contains

Scheme 1 Schematic illustration of the applications of cholesterol (a smallbiomolecule found in biological cell membranes (top left)) in the field of biona-notechnology: self-assembly of cholesterol-containing polymers into nano-structures e.g. polymersomes (top right), cholesterol as a constituent of liposomes(bottom right), and cholesterol as an anchoring unit for liposomes e.g. for cap-sosome assembly (bottom left).


both a polar hydroxyl group on one side and a non-polar tet-racylic steroidal ring structure with a branched hydrocarbon tailon the other (Fig. 1a). Further, cholesterol is comprised of arigid planar subunit due to the trans-conguration of the ringsand a exible iso-octyl side chain ‘tail’. The combination of thisrigidity and exibility gives cholesterol the property of orienta-tional ordering and display of liquid crystalline behaviour incombination with amphiphilic lipid membrane bindingcapabilities.

Conjugation of cholesterol

The majority of conjugation strategies used to conjugatecholesterol onto molecules, biomolecules, or macromoleculesof interest rely on the hydroxyl group in the 30 position of thecholesterol ‘A’ ring (Fig. 1b). The most common are esterica-tion, carbamate, and carbonate bond formation usingchloroformate derivatives. These strategies allow commoncarboxylic acids, amines, or hydroxyl functional groups whichare found on the most biologically important or other relevantmolecules, to be coupled onto cholesterol. Other strategiesapplied to conjugate to cholesterol are illustrated in Scheme 2and discussed in the next paragraphs.

Of further interest, Achalkumar et al. have recently reviewedthe chemistry and approaches used to make cholesterolconjugate linkers for tethering onto phospholipid bilayers.45

A practical challenge with any conjugation strategy used forobtaining amphiphiles based on cholesterol is the choice ofsolubilising conditions. The challenge is to nd a solvent thatwill dissolve both the hydrophobic cholesterol, and, at the sametime, the hydrophilic material onto which it is conjugated.Typical solvent choices range from chloroform, dichloro-methane, and dioxane, to tetrahydrofuran (THF). Furthermore,

Fig. 1 (a) A structural depiction of a cholesterol molecule highlighting the fourdomains of functional importance ((A) important for polarity and hydrogenbonding, (B) sterol rings, its conformation is important as it provides a rigid planarskeleton, (C) controls the conformation of the side chain, and (D) branchedaliphatic side chain) (left) to function in membranes as a flat elongatedcompound. The structure presumed to form in cellular membranes (right).Reprinted with permission from ref. 37. Copyright (2011) American ChemicalSociety. (b) Cholesterol molecule showing the tetracyclic steroid frame (A–D) withnumbering of carbon atoms of the sterol nucleus and side chain based on theconventional numbering system.17,254

Nanoscale, 2013, 5, 89–109 | 91


Scheme 2 Schematic illustration of synthetic opportunities and conjugationstrategies to the cholesterol alcohol. Clockwise from the top: etherificationthrough the trichloroacetimidate approach and via Williamson’s ether synthesis,dioxaphospholane cyclic phosphate functionalisation, carbamate and carbonateconjugation through a chloroformate, and esterification.


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derivatives based on cholesterol oen have a strong tendency toself-assemble. This oen complicates chemistry isolationprocedures such as water washes during solvent extractionsteps. Nonetheless, chemical synthetic procedures for couplingcholesterol are well represented in the literature to indicate thatthese issues can be overcome.

Chloroformate

A common strategy for cholesterol conjugation is the use of thechloroformates. Cholesterol chloroformate, a commerciallyavailable compound, is simply combined with thematerial to beconjugated onto, which carries an amine, to give a carbamatecholesterol derivative. The addition of the components isusually performed under cooled anhydrous conditions and inthe presence of a catalytic amount of a base like triethylamine or4-dimethylaminopyridine (DMAP). This strategy has been usedfor the coupling of cholesterol onto, for example, the primaryamines of 30-O-(6-aminohexyl)-uridine46 or polyethylenimine(PEI).47 Also commonly applied, alcohols can be coupled tocholesterol chloroformate to give the corresponding carbonate.As an example, the alcohol of hydroxylethyl (meth)acrylate(HEMA) was coupled to cholesterol, as a way of placing a shortspacer between the cholesterol and the vinyl group.48

Fig. 2 Schematic examples of cholesteryl-functional polymers: (a) bis-cholesterylend-functional P(HPMA),57 (b) ABA triblock copolymer (PNAM-b-PChA-b-PNAM),94 and (c) poly(amidoamine)-co-(amidoamine disulfide cholesteryl)copolymer.102

Esterication

The hydroxyl group lends itself well for esterication reactionsand, as such, several approaches have been used. Acylation ofthe hydroxyl moiety of cholesterol with commercially available,functional acid chlorides, such as acryloyl chloride49,50 and

92 | Nanoscale, 2013, 5, 89–109

methacryloyl chloride,51–53 allow appropriate vinyl esters to besynthesised in high yields. Further, the acid chloride can beobtained via reaction of the appropriate carboxylic acid withthionyl or oxalyl chloride prior to the reaction with choles-terol.54,55 Acid bromides, such as (2-bromoisobutyryl bromide)can also be efficiently coupled onto.56 Steglich estericationusing N,N0-dicyclohexylcarbodiimide and DMAP, has been usedsuccessfully to react a carboxylic acid with the alcohol ofcholesterol (Fig. 2a).57,58 Ring opening of succinic anhydridewith cholesterol can also be used to form a cholesterol esterwith a terminal carboxylic acid which can then be used forfurther chemical attachments.59

Etherication

Ether formation from the hydroxyl group is a common strategyfor the attachment of PEG and sugars to cholesterol. Forexample, cholesterol functionalised monomers containing adiethyleneglycol60 spacer and tetraethylene glycol10 spacer,attached via an ether bond, have been synthesised. Williamsonether synthesis is also a popular synthetic approach which can




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be carried out by reaction of the alkoxide of the alcohol with analkylating agent carrying the ‘R’ group. Alternatively, tocombine two alcohols together, one of them can be initiallytosylated (using p-toluene sulfonyl chloride) and allowed toreact with the other alcohol to form the corresponding ether.This was successfully carried out for the synthesis of a norbor-nene based monomer61 and a vinyl monomer containing analiphatic spacer.62

A glycolipid library based on acylated cholesteryl-b-galacto-sides was synthesised for the elucidation and construction of avaccine against Lyme disease. Here, the glycosylation ofcholesterol with galactose, essentially an ether bond formation,was achieved using a trichloroacetimidate approach.63 Several3-cholesteryl 6-(glycosylthio)hexyl ether glycolipids were alsoprepared via thiol alkylation using 3-cholesteryl 6-iodohexylether. The latter was formed from cholesteryl p-toluenesulfo-nate via three steps.64

Miscellaneous chemistry

An example of the cyclic phosphate functionalisation ofcholesterol was published by Iwasaki and Akiyoshi as a cho-lesteryl-functional monomer strategy for the synthesis ofbiodegradable and biocompatible polyphosphates.65 Thesynthesis of this monomer was achieved by the addition ofcholesterol to a cooled solution of 2-chloro-2-oxo-1,3,2-dioxa-phospholane in ether. However, only a 33% yield was achievedaer recrystallisation.

Monomers and polymers

The conjugation techniques previously mentioned arecommonly applied to the synthesis of cholesterol-based (cho-lesteryl) monomers for the synthesis of polymers. Esterica-tions and carbamate formation are typically used to add theappropriate polymerisable or polymer initiating group tocholesterol. These can then be used to create a diversity ofpolymer architectures such as end-functional polymers,homopolymers, copolymers and block copolymers.

Polymer end-functionalisation

Ring opening polymerisation (ROP)66 is one approach that hasbeen used for adding efficiently cholesteryl end-functionality topolymers. The polymerisation is simply initiated by an alcohol, inthis case cholesterol, which conveniently ring opens the mono-mers without the use of a catalyst. This approach has been usedto synthesise polyesters, polylactide (PLA), polyglycolide and theircopolymers, poly(lactide-co-glycolide) (PLGA)67 and, with theaddition of a catalyst Sn(Oct)2, 3-caprolactone, which ring opensto give poly(3-caprolactone).68 Polycarbonates based on tri-methylene carbonate69 or 2,2-dimethyltrimethylene carbonate70

were also easily synthesised without the use of a catalyst.Klok et al. used ROP to great effect to synthesise L-lactic acid

oligomer linkers initiated by cholesterol and terminated bycholesterol, or bioactive drugs, uorescent markers,71 andgeneration 1, 2 and 3 L-lysine dendrons.72

End-functionalisation of a radical polymer with acholesteryl moiety can be performed simply by the use of a


cholesteryl-functional initiator. An example is the use of a cho-lesteryl radical initiator in conjunction with a cholesteryl-func-tional vinyl monomer. In this case 4,4-azobis(4-cyano-1-cholesteryl) pentanoate azo initiator was used to initiate thepolymerisation of sodium 2-(acrylamido)-2-methylpropanesulfo-nate to give a cholesteryl end-capped and linear poly(sodium 2-(acrylamido)-2-methylpropanesulfonate).54 In another report,poly[N-(2-hydroxypropyl)methacrylamide]s (PHPMAs) with aterminal cholesteryl moiety either at one or both ends of thepolymer chains have been synthesised (PHPMA-Chol andPHPMA-2-Chol). These were prepared by the radical polymerisa-tion of N-(2-hydroxypropyl)methacrylamide, initiated with 4,40-azobis-[(3-cholesteryl) 4-cyanopentanoate] in the presence of2-mercaptoethanol and thiocholesterol chain-transfer reagents,respectively.73

A cholesteryl functional reversible addition fragmentationchain transfer (RAFT)74–78 agent with a single cholesteryl func-tionality placed in the RAFT re-initiating the ‘R’ group was usedfor the synthesis of u-cholesteryl functional poly(polyethyleneglycol) acrylate.58 A bis-cholesteryl functional ‘R’ group RAFTagent was used to place two cholesteryl u-end groups ontopoly(N,N-dimethyl acrylamide) (PDMA), PHPMA and copoly-mers thereof.57

Atom transfer radical polymerisation (ATRP)79–81 has alsobeen used as a tool to initiate polymerisations with a choles-teryl-functional initiator.82 Xu et al. applied this technique tosynthesise a biomimetic amphiphile from 2-(methacryloyloxy)ethyl phosphorylcholine from 10-cholesteryloxydecanyl-2-bro-moisobutyrate.83,84 Similarly, cholesteryl-2-bromoisobutyratewas used to initiate various oligo(ethylene glycol) (meth)acry-lates to produce copolymers with u-bromine end-groups. Thesebromine end-groups were later transformed to reactive azidesby nucleophilic substitution with sodium azide and, onceassembled into micelles, allowed the azide–alkyne cycloaddi-tion “click” reaction to occur in the corona of the micelle.56

Post-functionalisation of end-groups

The exibility of RAFT agents allows for a symmetrical design ofpolymer end-group functionality via the RAFT agent. This canbe achieved by using a RAFT agent that has a central bis-func-tional radical leaving/reinitiating the ‘R’ group and two thio-carbonylthio ‘Z’ groups on either side. Post-functionalisationcan then be carried out through the aminolysis of the thio-carbonylthio end-groups which transform them into thiols.Such an approach has been used to react end group thiols with3-cholesteryl 6-iodohexyl ether via nucleophilic reaction tomake a,u-di(cholest-5-en-3b-yl-6-oxyhexylthio) poly(N-isopropylacrylamide) P(NiPAM).85 A pyridyldisulde poly(oligo (ethyl-eneglycol) acrylate) RAFT polymer with a pre-attached bovineserum albumin (BSA), was functionalised with a cholesterylgroup through disulde exchange with thiocholesterol.86

Homopolymers

One of the ways of introducing several cholesteryl functionalitiesalong the length of a polymer chain is by homopolymerising acholesteryl-functional monomer. Ruthenium-catalysed ring

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opening metathesis polymerisation (ROMP)87,88 of a monomerfunctionalised with both norbornyl and cholesteryl moieties hasbeen utilised to make pendant cholesteryl-functional homopoly-mers with a variety of spacers present between the backbone andthe cholesterol.55

A technique that has been used more prolically for intro-ducing cholesteryl-functionality along a polymer chain is theradical polymerisation of cholesteryl-functionalised acrylate ormethacrylate monomers to give pendant cholesteryl function-ality to polymers. This was looked at as early as the late 60’s and70’s by Hardy et al.89,90 and De Visser et al.49,50 More recently,cholesteryl polymerisation has seen resurgence when pairedwith controlled radical techniques like ATRP and RAFT. Forexample, a homopolymer based on cholesteryl acrylate (CA)using RAFT was synthesised and block extended with styrene.91

Block copolymers

Zhang et al. designed cholesterol monomers with a hydrophilictetra(ethylene glycol) (TEG) spacer inserted between the meso-gen and the acrylate (or methacrylate) polymerisable group inorder to increase the water solubility of the monomer in theirdispersion polymerisation system.10 From the poly(methacrylicacid (MAA)-co-PEG methacrylate (PEGMA)) copolymer macro-RAFT agent, they synthesised the block copolymer P(MAA-co-PEGMA)-b-P(Chol-TEGMA) under dispersion polymerisationconditions, which allowed the formation of a block copolymervia self-assembly in minimal steps. In a different report, Liuet al. used a diethyleneglycol spacer between the cholesterol andthe acrylate and made blocks with (benzyl protected) ascorbatesections.92 In our own research, we designed an amphiphilicblock copolymer poly(N-vinyl pyrrolidone) P(NVP)-b-P(CA)which was obtained by polymerising a short CA oligomersection of, on average, 3 units from a P(NVP) macroRAFTagent.93 This macroRAFT agent was obtained via the controlledpolymerisation of NVP with a xanthate RAFT agent. Alterna-tively, diblock copolymers have also been designed by growingthe second block from a cholesteryl methacrylate (CMA) mac-roRAFT agent with a trimethylsilyl protected hydroxyethylmethacrylate (HEMA-TMS), giving P(CMA-b-HEMA-TMS). Thisblock copolymer was further deprotected by the addition ofconcentrated HCl in a THF solution resulting in the cleanproduct copolymer P(CMA-b-HEMA).51 Curiously, the authorsreported problems with controlling of the polymerisation of theunprotected HEMA. Additionally, they initially observed diffi-culties polymerising CMA under ATRP conditions with methyl2-bromopropionate as the initiator and copper(I) chloride asbipyridine as the catalyst–ligand system at 90 �C. Recently, wehave looked at the synthesis of an ABA triblock copolymer in twosteps from a bis ‘Z’ functional RAFT agent to form a CA mac-roRAFT agent, from which two N-acryloyl morpholine (NAM)arms can be grown (Fig. 2b).94 We designed the NAM : CA blockratio to be close to 2.5 : 1 to direct the spatial self-organisationof the polymer into polymersomes. A bis ‘R’ RAFT agent for thedesign of an ABA triblock with the cholesteryl as the ‘B’ block,for use in liquid crystalline block copolymers, has also beenlooked at by Shibaev et al.95

94 | Nanoscale, 2013, 5, 89–109

ROMP can also be successfully used to synthesise wellcontrolled block copolymers containing a cholesterol richsection. Here, a norborene functional cholesterol monomer ispolymerised to form the second block from a poly(ethyleneoxide) brush block. These block copolymers are assembled intonanoparticles for the delivery of doxorubicin and indometh-acin, through acid labile carbamate linkers.61

Random or statistical copolymers

Copolymerisation is a convenient method for the introductionof cholesteryl pendants into the polymer chain at various molepercentages. Some examples include the free radical copoly-merisation of acrylic acid (AA) with 5-acryloyloxypentyl choles-terate (Ch5A) to give P(Ch5A-co-AA),96 NiPAM with CA to giveP(NiPAM-co-CA),97 HPMA terpolymerised with 6-meth-acrylamido hexanoyl hydrazine and cholest-5en-3b-yl 6-meth-acrylamido hexanoate,98 and our own work via RAFT mediatedcopolymerisation to give P(MAA-co-CMA) with a 7 mol%cholesterol content.52,99 Researchers at the Australian Centre forNanomedicine have also recently looked at the copolymerisa-tion of CMA with MAA.100 They varied the CMA content from2 mol% to 8 mol% and together with dimethylamino ethylmethacrylates53 and RAFT mediated control, they were able toachieve a CMA content ranging from 2 mol% to 20 mol% whilstkeeping their polymer molecular weights around 20–24 kDawith low molecular weight dispersities throughout. Theamphiphilic copolymers showed pH-induced phase transitionsin aqueous media and are envisioned to be useful forcondensing and transporting siRNA due to their lipidmembrane destabilizing properties.

An example of a polyphosphate terpolymer containingpendant cholesteryl functionality, pendant ATRP initiatinggroups as well as 2-isopropyl-2-oxo-1,3,2-dioxaphospholane unitswere synthesised as a biodegradable gra terpolymer, a novelamphiphilic biomaterial which can function as a nanocarrier.65

Alternatively, post-functionalisation of polymers to introducecholesterol has been performed on polymers containing amine oracid reactive groups, PEI,47 poly(L-lysine) (PLL)52 and sodiumalginate,101 giving random copolymers. An interesting example ofpost-functionalisation is the Michael addition polymer poly-(amidoamine) (PAA) (Fig. 2c).102 The nanoparticulate polymernetwork can be obtained using cystamine as a cross-linking agentwhich can be made to react with 2,20-dithiodipyridine turningthem into linear PAAs with dithiopyridyl side groups that are ableto undergo a thiol exchange reaction with the commerciallyavailable thiocholesterol. The resultant products are examples ofamphiphilic PAA–cholesterol conjugates in which lipophiliccholesterol moieties are linked to the hydrophilic PAA chain bydisulde bonds. These are relatively stable in blood but arecleavable under the reductive conditions within cells.

There are many more examples of cholesterol containingpolymers, mostly with a focus on optical, liquid crystal andsome general bioapplications; however, these are beyond thescope of this review. The reader is directed to reviews by Zhouet al. and Yusa for a more exhaustive list and description ofthese polymers.103,104



Fig. 3 (a) Sequence and relative stability of metastable intermediates plotted asa functions of time after supersaturation of bile. Reproduced with permissionfrom ref. 105. Copyright (1994) National Academy of Sciences, USA. (b) Images ofa helical cholesterol ribbon. Phase images for angle of 0� illumination (i) and foraperture synthesis (ii). Reproduced with permission from ref. 113.


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Self-assembly of cholesterol

As previously mentioned, cholesterol is structurally composedof different sections which play a role in its interesting self-assembly behaviour in a variety of multi-componentsolutions.105–108

Helical ribbons were rst discovered in human gallbladderbile, and it was found that these structures form as a precursorof gallstones upon dilution of bile.107 Konikoff et al. conrmedthat cholesterol is the major constituent of these helical ribbonsby various experimental techniques including density gradientcentrifugation or X-ray diffraction. They also found that thehelical ribbons themselves are precursors of stable cholesterolmonohydrate crystals.106,107 Chung et al. used several model bilesystems which consisted of a mixture of three types of chiralmolecules in water: a bile salt, a phosphatidylcholine, andcholesterol. They demonstrated that helical ribbons formedwith two distinctive pitch angles, 11.1 � 0.5� and 53.7 � 0.8�.105

Zastavker et al. then showed that the self-assembly of the helicalribbons with these two distinct pitch angles was not unique tomodel bile, but was a general phenomenon for a variety of four-component systems composed of a bile salt or surfactants, aphosphatidylcholine or fatty acids, a steroid analogue ofcholesterol, and water.109 It was found that in undiluted solu-tions containing cholesterol, surfactants, and fatty acids orphospholipids, cholesterol is solubilised by micelles, i.e.cholesterol was contained in the core. These solutions containboth the free surfactants and the surfactants contained inmicelles in equilibrium. Upon dilution, the equilibrium shisfrom primarily micelles to primarily free surfactants. Themicelles break up and release the cholesterol, causing thesolution to become supersaturated with cholesterol. Subse-quent dilution leads to the formation of large cholesterolmonohydrate crystals. However, the exact structures that areformed and the sequence in which they form depend on thecomposition of the solution and the concentration of itscomponents as shown in Fig. 3a.105

In an attempt to assess the properties of helical ribbons,Starostin and coworkers studied the non-monotonic force-extension properties and predicted hysteresis behaviour for low-pitch ribbons of arbitrary material properties using a newmodel for inextensible elastic strips. It explained the rst-ordertransition between two different helical states as experimentallyobserved. Furthermore, they revealed a new uncoiling scenarioin which a ribbon of very low pitch shears under tension andsuccessively releases a sequence of almost planar loops. Theyconsidered their results to be relevant for nanoscale devicessuch as force probes.110 Also, helical ribbons were thought tohave potential serving as mesoscopic springs to measure or toexert forces on nanoscale biological objects. The springconstants of these helices depend on their submicroscopicthickness. Using quantitative phase microscopy, Khaykovichet al. discovered a quadratic relationship between the radius Rand the thickness t of helical ribbons that form spontaneouslyin multicomponent cholesterol–surfactant mixtures. Thequadratic relationship (R f t2) between radius and thickness isa consequence of the crystal structure of the ribbons and


enables the determination of the spring constant of any helicessolely in terms of its observable geometrical dimensions.111

Choi et al. used high-speed synthetic aperture microscopy toimage helical cholesterol ribbons in surfactant mixtures, as wellas to measure the thickness of a nanoscale cholesterol helicalribbon (Fig. 3b) which was determined to be 68 nm.112,113 Thesefundamental ndings provide the foundation towards applica-tions of the helical ribbons.

Self-assembly of cholesterol-modifiedpolymers into nanostructures

Due to its highly hydrophobic sterol skeleton, good biode-gradability and biocompatibility, hydrophobic cholesterol is agood choice for the amphiphilic modication of many water-soluble polymers. Self-assembly of amphiphilic cholesterol-modied polymers has been a subject of great interest for thepast few decades.71,73,101,114–116 Cholesterol units attached ontomacromolecules as pendant(s) or as end-group(s) can induceformation of structures of well-dened morphology in water asa result of specic interactions among the cholesterol moie-ties.103,104 Though cholesterol has been used to modify manykinds of polymers,84,102,117–120 herein we will focus on cholesterol-modied polymers and their self-assembly behaviour withpotential biomedical applications, e.g. drug delivery.

Cholesterol-modied polysaccharides have attracted theinterest of many researchers. Akiyoshi et al. prepared choles-teryl-bearing mannans (CHM) and cholesteryl pullulan (CHP).CHM and CHP can self-organise into nanoparticles and gels in

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Fig. 4 (i) Molecular structure of the diblock copolymer PEG-b-PAChol. (ii) Self-assembled structures of PEG45-b-PAChol in dioxane/water: cryo-transmissionelectron microscopy (TEM) image of vesicles of PEG45-b-PAChol10 (left) and cryo-TEM images of vesicles of PEG45-b-PAChol16 (right). (iii) Schematic presentation ofthe model for ellipsoidal smectic polymer vesicle (top) and smectic rod-likemicelles (bottom). Reproduced with permission from ref. 135.


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semi-dilute solution due to the hydrophobic interaction of thecholesterol moieties.121 Cholesterol-modied chitosan (CS-CH)can form self-aggregated nanoparticles in physiological saline.The drug-loading and release behaviour of the CS-CH nano-particles were investigated using Cyclosporine A (CyA) as amodel drug. CyA was physically entrapped in the nanoparticlesduring the dialysis process, and the drug-loading was on theorder of 6.2 wt% of the CS-CH self-aggregated nanoparticles.122

Yu et al. investigated cholesterol-modied glycol chitosan(CHGC), and used the CHGC self-aggregated nanoparticles fordoxorubicin loading and delivery.123 They found that doxoru-bicin release from CHGC nanoparticles was dependent on thepH, with faster release at lower pH. This aspect was consideredimportant, because the drug would be more efficiently releasedin the tumour tissue than in circulation. They reported that thepharmacokinetic parameters of doxorubicin changed in miceand more efficient tumor growth inhibition for doxorubicin-loaded CHGC compared to free doxorubicin was reported,making these nanoparticles promising carriers for insolubleanticancer drugs in cancer therapy. The Zhang group publisheda series of reports involving CS-CH124–128 and synthesisedcholesterol-modied O-carboxymethyl chitosan (CCMC),cholesterol-modied chitosan conjugate with succinyl linkages(CHCS) and 6-O-cholesterol modied chitosan nanoparticles(O-CHCS NPs). The self-aggregation behaviour of the modiedchitosan and chitosan nanoparticles due to the strong hydro-phobic interaction of the cholesterol moieties was investigated.It was found that the degrees of substitution (DS) of thecholesterol moieties played an important role in the size andthe surface property of the self-aggregated nanoparticles.Comparing the morphology and the stability of self-aggregatednanoparticles between CCMC and CHCS, the negatively chargedcarboxymethyl groups are advantageous for the formation ofwell-shaped and stable self-aggregated nanoparticles.126 Theystudied the drug loading and release property of CHCS andCCMC nanoparticles using Epirubicin (EPB) and paclitaxel,respectively.124,127 The results showed that the release ratedecreased with increasing pH of the media. In PBS, the EPBrelease was very slow (�24.9% in 48 h), while paclitaxel wascontinuously released over 84 h at 37 �C. The biodistribution ofpaclitaxel-loaded CCMC self-assembled nanoparticles showedthat these nanoparticles seemed to signicantly increase theuptake of paclitaxel in plasma, liver and spleen, but decreasedthe uptake in heart and kidney. Further, the interactionbetween BSA and self-aggregated CCMC and O-CHCS NPs withdifferent DS of cholesterol moieties was assessed.125,129 Theaddition of O-CHCS NPs led to the decrease of the a-helicalcontent of BSA and the increase of the b-strand content. Theunfolding of BSA by a denaturant such as urea was largelysuppressed upon interaction with CCMC self-aggregatednanoparticles.

Cholesterol is oen used to modify PEG polymers leading todifferent self-assembly behaviour.130–137 Jia et al. synthesisedamphiphilic liquid crystal block copolymers consisting of acholesterol-based smectic liquid crystalline polymer block(PAChol) and a PEG block (Fig. 4i).135 They found that PEG45-b-PAChol and PEG114-b-PAChol (45 and 114 are the degrees of

96 | Nanoscale, 2013, 5, 89–109

polymerisation of the PEG blocks) with different hydrophilic/hydrophobic weight ratios could self-assemble into polymeraggregates with different morphologies in THF/water anddioxane/water (Fig. 4ii). Adding water to a dilute solution ofcopolymers in dioxane yielded smectic polymer vesicles and/ornanobers. By using THF instead, solid spherical aggregates wereobtained upon water addition for the PEG45-b-PAChol copol-ymer. With proper block sizes and the use of appropriate organicco-solvents, these biocompatible cholesterol-based copolymerscan self-assembly into aggregates with desirable shapes (e.g.vesicles or nanobers) (Fig. 4iii), which have potential applica-tions in drug delivery and material science.135 Nagahama et al.prepared a novel star-shaped copolymer derivative with choles-terol end groups, cholesterol-substituted 8-arm PEG-b-poly(L-lac-tide) (8-arm PEG-b-PLA-cholesterol). Based on the specicself-assembly between the cholesterol end groups, the 8-arm



Fig. 5 Schematic representation of a polymersome (i) assembled from an ABAtriblock copolymer consisting of poly(N-acryloyl morpholine) and poly(cholesterylacrylate) with entrapped cargo in the hydrophobic part of its polymer membrane(ii), and a cryo-TEM image confirming the assembly of polymersomes (iii).Reproduced with permission from ref. 94.


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PEG-b-PLA-cholesterol aqueous solution exhibited instantaneoustemperature-induced gelation at 34 �C.131 In addition, PEGylatedpoly(N-methyldietheneamine sebacate)-co-((cholesteryl oxocarbo-nylamido ethyl) methyl bis(ethylene) ammonium bromide)sebacate) (PEG-P(MDS-co-CES)) were synthesised, which couldself-assemble into stable micelles of small size with a hydro-phobic core and hydrophilic shell.136,137 Different cholesterol-graing degrees of the PEG-P(MDS-co-CES) polymer were shownto have improved stability of micelle/DNA complexes in blood forsystemic in vivo gene delivery. In another report, Manakker et al.investigated the rheological properties of a self-assembledhydrogel system composed of b-cyclodextrin (bCD)- and choles-terol-derived 8-arm star-shaped PEG.133 They found that the 8-armpolymer-based mixtures yielded tight viscoelastic networks, buttheir storage and loss moduli signicantly deviated from thosepredicted by the Maxwell model. Rheological analysis alsoshowed that the hydrogels were thermo-reversible. At lowtemperatures, the gels showed viscoelastic behaviour due to slowoverall relaxation of the polymer chains. At higher temperatures,however, a reduced number of bCD–cholesterol complexes andconcomitant faster chain relaxation processes eventually led toliquid-like behaviour.

Cholesterol-modied compounds can also be mixed withothers, and self-assembled into different macroscopic vesicles.For example, the nucleoside 20-deoxy-20-aminouridine wasmodied with cholesterol, and the lipophilic nucleoside 20-N-(2-(cholesteryl)-succinyl)-20-deoxy-20-aminouridine was obtained.When mixed with the unsaturated phospholipid dio-leoylphosphatidylcholine, microtubes were formed. Fromconfocal uorescence microscopy images, the tubes appearedas open-ended cylindrical structures with outer diametersbetween 2 and 3 mm and lengths between 20 and 40 mm.138 Inanother report, the dialysis of a dimethyloxide solution ofcholesterol-modied dextran (Chol-Dex) and PLA against wateryielded hollow polymer nanocapsules with a highly stablestructure and relatively narrow size distribution.139 The hollowcapsules were thought to be formed by anchoring of most of theamphiphilic polysaccharide onto the surface of swelled aggre-gates as a result of phase separation of amphiphilic Chol-Dexfrom hydrophobic PLA. The capsules obtained have potentialapplication as drug carriers or as biomembrane models.

We recently reported the self-assembly of an amphiphilicblock copolymers containing cholesterol as the hydrophobicblock into polymersomes (Fig. 5).94 We synthesised an ABAtriblock copolymer with the ‘A’ segment consisting of poly(NAM),a non-ionic hydrophilic polymer with low-fouling propertiessimilar to PEG, and the ‘B’ segment of poly(CA) as the hydro-phobic part of the triblock copolymer. We demonstrated thestable, non-aggregated assembly of polymersomes with narrowpolydispersity. We used these polymersomes as reservoirs inpoly(vinyl alcohol) composite hydrogels and showed a signicantdecrease in viability of adhering myoblast cells when a cytotoxiccompound was entrapped in the polymersomes, thus conrmingthe potential of this system in surface-mediated drug delivery, abiomedical approach which aims to deliver therapeuticcompounds from the surface of an implantable device or a tissueculturing scaffold to adhering cells or to the surrounding media.


In summary, based on the hydrophobic interaction, choles-terol-modied polymers can self-assemble into differentmicelles, nanoparticles, nanotubes, nanogels, or polymer-somes. The size, shape and mechanical properties of theseassemblies are designed for encapsulation, storage, release anddelivery of therapeutics with potential application in biomate-rials and biomedicine.

Cholesterol and liposomes

The nal part of this feature article reviews the benets ofcholesterol incorporated into liposomes for biomedical appli-cations. Two different aspects of cholesterol, namely its abilityto act as stabiliser for lipid membranes and its ability to linklipid and polymeric structures together, are discussed. Exam-ples of applications in drug delivery and biosensing will beoutlined for both aspects.

Cholesterol as a stabiliser

Liposomes are among the prominent candidates for variousbiomedical applications in bionanotechnology. Liposomes arespherical vesicles which consist of one or more lipid bilayermembrane(s), encapsulating a volume of aqueous medium(Fig. 6a).140 Glycerophospholipids, sphingolipids, and choles-terol are the predominant components used for their prepara-tion. Their structural similarity to natural cell membranes,141

and their ability to mimic the behaviour of biological cellmembranes make liposomes useful in various applicationsranging from drug delivery,142,143 cosmetic,144 and foodscience145 to agriculture.145 However, control over the lipidbilayer permeability and stability towards solutes or biomole-cules is oen a challenge and can compromise their applica-tions in the aforementioned elds. This aspect can be regulatedby adjusting the cholesterol content of the lipid bilayer,146,147

since cholesterol affects the physicochemical properties of the

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Fig. 6 Cholesterol as stabiliser: (a) schematic illustration of the fundamental self-assembly process from individual phospholipid molecules (i) to bilayer membraneleaflets (ii), followed by transformation into liposomes (iii). A single bilayer consistsof arranged individual lipid molecules with their hydrophobic tails facing eachother and their hydrophilic head-groups facing toward the internal and externalaqueous media (iv). Adapted with permission from ref. 140. (b) Schematicrepresentation of liposomes with encapsulated enzymes. Substrates diffuse intothe void of the liposome through porin channels, react with the enzymes, and theproduct is released back into the solution through diffusion. Reproduced withpermission from ref. 141. (c) Schematic representation of a biosensor assay wherea DNA capture probe is immobilised on a polyethersulfone membrane and a DNAreporter probe is coupled to the surface of a liposome. When a specific E. colimRNA is present, a sandwich is formed between the capture probe, the E. colimRNA and the reporter probe, capturing the liposome in the capture/detectionzone. The number of liposomes is directly proportional to the amount of E. colimRNA present. Reproduced with permission from ref. 203. (d) Table containingmost common homogeneous complement mediated liposome immunoassays.Reprinted with permission from ref. 205. (ab, antibody; ag, antigen; BSA, bovineserum albumin; CF, carboxyfluorescein; chol., cholesterol; DCP, dicetylphosphate;DMPC, dimyristoylphosphatidylcholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-(10-rac-glycerol); DNP, dinitripheno(y)l(ated); DPPC, dipalmitoylphos-phatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine; DSPC, dis-tearoylphosphatidylcholine; EA, egg albumin; HSA, human serum albumin; PA,palmitic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SA, stear-ylamine; and SPH, sphingomyeline).


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lipid membrane. For an in-depth discussion, the reader isreferred to the reviews by Ohvo-Rekila et al. or Rog et al.148,149

The amphiphilic cholesterol is arranged in the lipid membraneby orienting the steroid ring parallel to the hydrocarbon chainsof the lipids (phospholipids or sphingolipids) and the hydroxylgroup encountering the aqueous phase. There are two physical

98 | Nanoscale, 2013, 5, 89–109

states of a lipid bilayer in the absence of cholesterol: the gelstate at lower temperatures and the liquid-crystalline state athigher temperatures. The individual lipid molecules of a lipidbilayer have temperature-dependent mobility (uidity). Alllipids have a characteristic temperature in which they undergo atransition from the gel to the liquid-crystalline phase, which isknown as phase transition temperature (Tm). The phasebehaviour of the lipid bilayer and thus Tm is determined by thevan der Waals (VDW) interactions between adjacent lipidmolecules. The interaction between the acyl chains of the lipidsis mainly governed by two factors: the length of the chain andthe packing of the lipids in the bilayer, i.e. longer tail lipids havemore area to interact, which will, in turn, increase the strengthof the interaction and, consequently, decrease the mobility ofthe lipid. The type of acyl chain (degree of saturation) plays animportant role in how the lipids bind in the bilayer, since anunsaturated double bond can produce a kink in the alkanechain which will create extra free space within the bilayer, thusallowing for additional exibility in the adjacent chains.Therefore, unsaturated lipids have a signicantly lower Tmcompared to saturated lipids. Incorporation of increasingamounts of cholesterol into the lipid bilayer induces an“intermediate” state by increasing and decreasing the uidity ofthe hydrocarbon chain below and above Tm, respectively, andeventually eliminating Tm entirely. In the gel phase, the pres-ence of cholesterol in the membrane weakens the VDW inter-actions between the hydrocarbon chains of the fatty acids andprevents lipid crystallisation, while in the liquid-crystallinephase, the hydrocarbon chains of the lipids which interact withthe rigid, planar rings of the cholesterol, are partly immobi-lised. Specically, cholesterol increases the degree of orienta-tion and reduces the rate of motion in the liquid-crystallinephase of the lipid membrane. This leads to a laterallycondensed membrane with increased packing density and, as aconsequence, higher mechanical strength and lower perme-ability. Nature makes use of this aspect in many ways, butnanotechnological applications also rely strongly on this effectas outlined in the subsequent paragraphs.

Drug delivery

Liposomes have been considered as drug delivery vehicles dueto their colloidal size, ease of preparation, controllable surfaceand membrane properties, large carrying capacity, andbiocompatibility for over 30 years.150 Since the liposomal shellcan enclose or bind many different classes of substances, lipo-somes are successfully employed as therapeutic agents for thedelivery of antibacterial,151 antiviral,152 and anticancer drugs,153

as well as hormones,154 enzymes,155 and nucleotides.153,156

However, two of the major drawbacks of liposomes as drugcarriers are their oen poor stability in blood circulation uponsystemic application and the sometimes suboptimal controlover cargo retention and release.157 In the latter case, drugs haveto be transported to their site of action quantitatively, or thetherapeutic cargo has to be gradually released.146,147

Cholesterol has served to overcome both of the above-mentioned challenges. Since the addition of cholesterol reduces




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the uidity of the lipid bilayer and increases the stability of theliposomal membrane,158 adjusting the cholesterol content ofliposomes has been one of the prime ways to control thepermeability to solutes both in vivo and in vitro. Typically, half-lives of passively encapsulated drugs range fromminutes, in eggphosphocholine liposomes, to a dozen hours for cholesterol-containing liposomes.159 Owing to this increased cargo reten-tion, the rst liposomal formulation of the antitumor drugDoxorubicin (Doxil�) was approved in 1995 by the Food andDrug Administration (FDA) for medical use in oncology.160,161

Other cholesterol-containing liposomal formulations of drugs,i.e. Daunorubicin (DaunoXome�) used for the treatment ofadvanced HIV-associated Kaposi’s sarcoma,162 Amphotericin B(AmBisome�, Amphotec�, and Ableet�) for the treatment offungal infections in febrile neutropenic patients as well as forthe treatment of aspergillosis, candidiasis, and cryptococcosisinfections refractory to Amphotericin B,163,164 and cytarabine(DepoCyt�) for the treatment of lymphomatous meningitis,165

were FDA approved in 1996 (DaunoXome�), 1997 (AmBiso-me�), and 1999 (DepoCyt�), respectively, and are still on themarket.164,166–168

Liposomal formulations containing cholesterol of differentdrugs, such as vincristine sulfate169 or cisplatin (Lipoplatin),170

have not been FDA approved yet but are on Phase II and PhaseIII clinical trials, respectively.

Analytical science

Interest in liposome technologies and applications for analyt-ical purposes is constantly growing, and four major areas ofactivity can be identied, namely liquid chromatography (LC),capillary electrophoresis (CE), biosensors, and immunoas-says.140 In all four analytical techniques, cholesterol plays animportant role in liposome stabilisation.

LIQUID CHROMATOGRAPHY. The understanding of the inter-action between cell membranes and different molecules (e.g.drugs, proteins, and peptides) is of great importance in pre-dicting the behaviour of the drug in the organism. In vivoinvestigations of the fraction of the drug adsorbed in humansand animals are time-consuming and difficult to perform. Sinceliposomes structurally closely resemble natural membranes,they are extensively used in medical and pharmaceuticalresearch as models for mimicking the structure and the func-tion of biological cell membranes.171,172 The use of liposomesimmobilised within the pores of carrier gel beads as a stationaryphase for column chromatography with an aqueous mobilephase is known as immobilised liposome chromatography(ILC),173 a method that emerged in the 1990s171 and has provensuccessful in studying solute–membrane interactions.174 In ILC,variations in the retention volume depend on the extent ofsolute–membrane interaction and can be precisely measured.175

Liposomes can be immobilised in these columns by severalways173,176–178 including avidin–biotin interaction.140,171,179 Thisspecic binding pioneered by Yang et al. allowed prolonging thecolumn lifetime for over a year.175,179,180 Further, these liposomecolumns have successfully been used to predict the drugabsorption in vivo by estimating the membrane partitioning


coefficient of the diverse drugs. The results showed that, fordrugs that follow the transcellular passive transport route, theliposome column could be used for a preliminary drugscreening, offering an alternative to the high throughput drugscreening system based on Caco-2 cells used by pharmaceuticalcompanies.175,181 In this context, cholesterol has been importantas a component of the small-intestine brush-border membraneswhich are composed of egg phosphatidylcholine, phosphati-dylserine (PS), phosphatidylethanolamine, and cholesterol.182

Liu et al. reported retention data of numerous drugs utilisingILC and liposomes made of these components to mimic theintestinal membranes. The aim was to predict drug intestinaladsorption, since absorption of orally administered drugs in theintestine (i.e. uptake of the drug through the cell membrane) isthe rst step of the drug’s action.175,181,183

CAPILLARY ELECTROPHORESIS. CE is an analytical techniquethat separates ions based on their electrophoretic mobility withthe use of an applied voltage. Liposome capillary electropho-resis (LCE) is a recent approach that involves the use of lipo-somes as coating material184,185 or carrier.171,172 The details of thetechnique are beyond of the scope of this review and for a moredetailed overview, the reader is referred to other reports.171,172

Here, just two examples to illustrate the importance ofcholesterol are discussed. Wiedmer et al. employed 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and POPC/PS lipo-somes with various amounts of cholesterol as carriers andsteroidal sex hormones as model analytes. The study investi-gated the inuence of cholesterol on the sizes and electropho-retic mobility of POPC and POPC/PS liposomes with specialfocus on the inuence of cholesterol on possible selectiveinteractions between the steroids and the liposomalmembranes. Their results showed that the presence of choles-terol slightly increased the liposomal diameter, and signicantdifferences in selectivity between the steroids and the liposomalmembranes were achieved through the addition of choles-terol.186 Alternatively, liposomes have been used as coatingmaterials in CE in order to mimic biological cell membranes,since cell membranes are mainly composed of phospholipidsand cholesterol, and the interaction between the liposomes andthe analytes has been studied.185,187 As an example, Hautala andcollaborators showed the coating of silica capillaries withanionic liposomes comprising POPC, bovine brain PS, andcholesterol, and evaluated the coating effectiveness by exam-ining the interactions of steroids with the coated capillaries.Their results show that the capillaries can be successfullycoated with liposomes and those coated capillaries can be usedto study the interactions between phospholipid bilayers anduncharged steroids. Since the steroids are neutral under theapplied conditions, the separation is based on hydrophobicinteractions with the liposome coating.187

BIOSENSORS. Biosensors show promise in many aspects ofmedical care from drug development over early detection ofdiseases to monitoring the progress of a treatment. All theseapplications require sensitive, rapid, cheap, and reproducibledetection and recent advances in nano- and biotechnology aswell as chemistry, biochemistry, physics, and biology havemadeoutstanding contributions141 for the development of biosensors

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in medicine,188–190 for environmental applications,191 and in thefood industry.192,193 However, ways to immobilise biologicalmolecules while preserving their activity remains one of themajor challenges in the development of advanced biosensors.Encapsulation techniques are among the most favourableapproaches to entrap biological molecules while preservingtheir functionality for an extended period of time. Variousapproaches have been employed for the encapsulation of bio-logical material including polymeric vesicles,194 biomolecule–nanoparticle hybrid systems,195 capsules assembled via theLayer-by-Layer (LbL) technique,196,197 or single-enzyme nano-particles where each enzyme molecule is surrounded by aporous composite organic/inorganic network.198,199 However,liposomes have emerged as lead candidates by providing acomfortable cage which can preserve the biomolecule activity ina three dimensional space, which is particular important forenzymes.141 Fig. 6b shows a representation of a liposomeencapsulating enzymes. The substrate can diffuse across themembrane through channel gates, react with the enzymes, andthe products can be released back in solution.141 In addition,encapsulation offers other benecial properties e.g. amplica-tion since liposomes concentrate the enzymes in a connedspace, thus, the enzymes are able to convert a higher amount ofsubstrate molecules into their corresponding products.172 Thisfeature is benecial in a number of biosensor applications suchas the determination of pH200 and oxygen,201 or the detection ofbacteria202,203 and other agents.204 For the creation of liposomesin biosensing applications, cholesterol plays a crucial role incontrolling the uidity and, in particular, the permeability ofthe lipid membrane in order to ensure stable entrapment ofmolecules required for detection.172

An interesting example of cholesterol-containing liposomesin this context was reported by McNamara and co-workers, whodeveloped a uorescence-based oxygen nanosensor by encap-sulating an oxygen-sensitive dye in the internal compartment ofthe liposomes.201 A major concern when developing liposomaluorescence-based sensors is leaking of the dye molecules fromthe supportive matrix. To this end, McNamara and collabora-tors prepared liposomes with a lipid composition consisting ofdimyristoylphosphatidylcholine, cholesterol, and dihexadecylphosphate and showed high stability toward dye leaking atroom temperature for 8 days, a fact which could be attributed tothe addition of cholesterol to the lipid membrane.

Since safety of water and food supplies has to be ensured toavoid contamination of infectious agents, the detection of path-ogenic organisms remains a crucial aspect. With the aim toaddress this issue, Baeumner et al. developed a nucleic acid basedsensor, also known as a genosensor or gene probe, which is ahighly sensitive and specic RNA biosensor for the rapid detec-tion of viable Escherichia coli (E. coli) as an indicator of pathogenicorganisms in water.203 Briey, the mRNA from E. coli was extrac-ted, puried, amplied, and then quantied with the biosensor.The biosensor is a membrane-based DNA/RNA hybridisationsystem and cholesterol-containing liposomes (a mixturecomposed of dipalmitoyl phosphatidyl choline (DPPC), choles-terol, dipalmitoyl phosphatidyl glycerol, and dipalmitoyl phos-phatidyl ethanolamine) encapsulating sulphorhodamine for

100 | Nanoscale, 2013, 5, 89–109

signal amplication. The method is based on the formation of asandwich between a DNA-capture probe immobilised on a poly-ethersulfonate membrane, the target mRNA sequence, and aDNA-reported probe coupled to the surface of a liposome (Fig. 6c).When a specic E. coli mRNA is present, a sandwich is formedbetween the DNA capture probe attached onto the poly-ethersulfonate membrane, the E. coli mRNA, and the reporterprobe attached to the liposomes, thus the liposomes beingcaptured in the capture/detection zone. Since the number ofliposomes is directly proportional to the amount of E. colimRNA,when non-specic RNA is present instead of E. coli mRNA, theliposomes are not captured in the capture/detection zone, and nosignal is reported in the detection zone.203

IMMUNOASSAYS. Immunoassay formats rely on a directly orindirectly detectable label. The label can be an easily detectablemolecule (e.g. radioactive or uorescent) or, alternatively, anenzyme. In an enzyme linked immunosorbent assay (ELISA) theenzyme label generates a measurable amount of product whichis (inversely) proportional to the unknown concentration ofanalyte (usually an antigen).205 The technique essentiallyrequires any ligating reagent which can be immobilised, adetection reagent that will bind specically, and an enzyme thatwill generate a signal that can be quantied. LISA, liposomeimmunosorbent assay, is the counterpart of ELISA, with theenzymes replaced by liposomes encapsulating a large amount ofmarker and receptor molecules. The sensitivity of the system ishigh and the additional advantage is that, by lysing the lipo-somes, the detectable markers are released immediately, thusavoiding the ELISA incubation step in which the substrate isconverted into the product.172 Similar to an ELISA, the encap-sulated marker can be colorimetrically, uorimetrically, chem-iluminometrically, or electrochemically detected upon lysis ofthe immobilised liposomes.205 Additionally, most formatsapplied in enzyme immunoassays can be used in combinationwith liposomes.205 The stability of the liposomes is of para-mount importance for successful LISA performance e.g. interms of sensitivity and selectivity. To increase the stability ofthe liposome membrane, cholesterol in combination withsaturated lipids has been employed.205

Fig. 6d depicts a table with the most commonly used lipo-somal compositions and the markers used to detect differentanalytes. As shown in the table, most liposomal compositionscontain cholesterol to equip the lipid bilayer with additionalstability.205 For instance, Kim and collaborators reported thecreation of a liposome immunoassay for the detection of genta-micin using phospholipase C to release the uorescent markerfrom the liposomes.206,207 Gentamicin, a drug frequently used forthe treatment of serious Gram-negative microbial infections, waschosen as a model analyte.206 The liposomes were composed of amixture of DPPC and cholesterol, since it was previouslydemonstrated that this composition allows the lysis of the lipo-somes by the lowest concentration of phospholipase C.206,207

Cholesterol as anchor

So far, the aspect of the cholesterol’s ability to affect themembrane properties of the liposomes and the consequences




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for different biomedical applications have been discussed. Thissection considers the use of cholesterol for a different purpose,namely as an anchor either to immobilise the liposomes to asurface for biosensing applications, or to coat liposomes withspecic molecules.

Biosensors

Liposomes hold enormous potential for biosensing applica-tions, not only as labels as discussed previously, but also for theestablishment of a successful platform for high-throughput(membrane) protein sensing.208 Immobilised liposomes in anarray format can act as supports for (membrane) proteins sincethe lipid bilayer provides a favorable environment that allowsprotein exibility and movement while avoiding their denatur-ation and loss of function. These sensors are of paramountimportance since proteins play a major role in disease devel-opments and 60–70% of the drug targets are (membrane)proteins.209

Approaches to immobilise intact liposomes include linkagessuch as avidin–biotin,210,211 interaction between antibody andantigen,212,213 disulde bonds,214 or the chemical linkagebetween histidines,215 but also the chemisorption of vesiclesonto gold lms.214 However, when these types of linkages areused, the controlled immobilisation of different types of lipo-somes to predened spots on the surface is impossible. Thischallenge has been addressed by tethering liposomes to solidsupports via DNA-directed coupling, an approach that was rstintroduced by Niemeyer and co-workers.216

Several strategies have been developed for the DNA-labelingof intact vesicles by using functionalised lipids such as thiol-reactive maleimido-lipids217,218 and thiol derivatives of DNA.219

Alternately, several approaches which make use of cholesterol-modied DNA for spontaneous anchoring of liposomes to(patterned) substrates with immobilized complementarystrands have been reported (Fig. 7ai).220–223 This non-covalentapproach is advantageous compared to the previouslymentioned concepts since it is largely independent of the lipidcomposition of the liposomes, does not require chemicalmodication of a lipid, has fast coupling rates, and there is no-need of introducing functionalised lipids. Therefore, by makinguse of a naturally occurring membrane constituent, unwantedside reactions between the functional groups and othermembrane constituents are eradicated. However, cholesterol-based anchoring of DNA is relatively weak, i.e. it is reversible. Inorder to allow tagging of a type of liposomes with a specic DNAsequence, the cholesterol anchor has to be strengthened byusing bivalent cholesterol DNA coupling.224 This approachallows sorting of red and green liposomes from a mixture(Fig. 7aii). In a continuation of this approach, ligand binding toliposome arrays equipped with G-protein coupled receptors hasbeen demonstrated on the way to develop this platform into aheterogeneous, self-sorting biosensing platform (Fig. 7b).225

PEGylation

One of the main drawbacks of liposomes in drug delivery istheir low stability in systemic circulation. Stealth liposomes


were created when it was realised that neither mechanical norelectrostatic stabilisation could improve their performancesignicantly enough.157 It has been widely demonstrated thatthe incorporation of glycolipids such as monosialogangliosideGM1

226 into the lipid bilayer or the coating of liposomes withhydrophilic polymer chains like polysaccharides226 or, prefer-entially, PEG226,227 becomes an efficient stabilisation tool byproviding a steric barrier. The particular efficiency of surface-attached PEG chains has been explained by the combination oftheir water solubility and exibility, which sterically stabilisesthe liposomes by preventing them from self-aggregation andfusion, as well as from opsonin adsorption, the rst steptowards clearance by the reticuloendothelial system.226–228 PEGcoated liposomes remain in the blood for up to 100� longerthan ordinary liposomes, thus increasing the pharmacologicalefficacy of the encapsulated agents,157 and passive targeting totumors has been reported.227 The reader is referred to recentreviews229–232 for a more detailed discussion on the topic ofPEGylated liposomes.

There are different ways to coat liposomes with PEG such asby graing it to the lipids by covalent bonding.226 Alternatively,cholesterol or phospholipids have been employed to anchorPEG to the liposome membrane, and it has been shown thatcholesterol-PEG was easily incorporated, making cholesterolthe more successful anchor moiety.104,158,226–228,233–235 They spec-ulated that long anchor units are difficult to insert into theliposomal membranes or that there is a higher affinity betweencholesterol and the constituent lipids.227 PEG-cholesterol hasthe added benet that it confers cohesion on lipid bilayers byinducing tighter molecular packing and restricting the motionsof the hydrocarbon chains, thus improving the cargo reten-tion.226 Long-circulating liposomes containing cholesterol–PEGhave already been successfully used in the cisplatin delivery invitro and in vivo.158 Fig. 7ci depicts a schematic illustration ofliposomes containing a mixture of cholesterol-PEG and thio-lated cholesterol for cisplatin encapsulation. The in vivocisplatin concentrations in the kidney tissue of Chinese KunMing mice aer injection of the liposome formulation and freecisplatin were compared (Fig. 7cii). The results showed thatPEGylated liposomes could successfully avoid the peak-concentration in kidney aroused from free cisplatin, reducingthe in vivo acute nephrotoxicity.158

Capsosomes

Sub-compartmentalised systems hold tremendous potential ascell mimics, which is an approach that aims to substitute forlost or missing cellular function.236 Articial multi-compart-ment systems do not need to possess the entire complexity ofbiological cells, but a specic key feature, which will mostlydepend on the application in mind (e.g. enzyme encapsulationfor enzyme replacement therapy to provide a biological-inspiredlong term solution for patients with chronic diseases).Mimicking the hierarchical sub-compartmentalised structure ofcells is essential to perform triggered, spatially separated,encapsulated (enzymatic cascade) reactions.52,237 Among thereported multi-compartment systems (e.g. vesosomes,238

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Fig. 7 Cholesterol as anchor: (a) (i) schematic illustration of a tethered vesicle–DNA assembly, showing how the hybridisation of complementary cholesterol–DNA pairs forms the (mobile) tether. The DNA array was produced by preferentialadsorption of biotin–BSA to Au spots, leaving the surrounding SiO2 substrateavailable for spontaneous supported phospholipid bilayer (SPB) formation, fol-lowed by the addition of NeutrAvidin, which binds to biotin–BSA-modified Auonly. Biotin–DNAB and chol–DNAA specifically binds to NeutrAvidin/biotin–BSA/Au and SPB/SiO2, respectively, followed by the immobilization of liposomestagged with the complementary DNA. (ii) Micrographs illustrating sorting ofliposomes tagged with different DNA. Micrographs (1) and (2) were obtained byexposing the DNA-modified substrate to a mixture of red-labelled and green-labelled liposomes tagged with different monovalent coupled chol–DNA. (3) and

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liposomes within a liposome carrier, polymersomes within apolymersome,239 polymer capsule within a polymercapsule,240,241 polymer hydrogel capsules containing cubo-somes242 or polymersomes243 and cellosomes,244 yeast cellsassociated with a polymer membrane)245,246 capsosomes, intactliposomes embedded into a polymer carrier capsule,52 are anovel, well-characterised system with potential in therapeuticcell mimicry. Capsosomes, as a dual composition system, cantake advantage of the different properties of the sub-compart-ments and the carrier capsule. Liposomes, due to the inherentlyhigh impermeability of their lipid membranes, are ideal toencapsulate small or fragile cargo. The polymeric carriers, dueto their semi-permeable nature, permit the interaction betweenthe internal and the external environment while providing thestructural integrity and help to overcome some of the limita-tions of liposomes such as instability under in vivo conditionsand the lack of control over their degradation. Capsosomes areassembled via the LbL technique,247 by alternatingly depositingpolymer and liposomes onto silica particles, followed by theassembly of the polymer carrier capsule and core dissolution(Fig. 8a).

With the aim of stably anchoring the liposomes into thepolymer membrane, we employed cholesterol-modied poly-mers, i.e. cholesterol-containing PLL (PLLc),52,99 poly-(methacrylic acid-co-cholesteryl methacrylate) (PMAc),52,99 andpoly(N-vinyl pyrrolidone-block-cholesteryl acrylate) (P(NVPc)).93

We also showed that PMAc was the more successful polymer toanchor liposomes than PMA containing oleic acid (PMAoa).248

Interestingly, while PMAc, PMAoa, and PLLc led to the formationof capsosomes with the liposomal units associated with thepolymer carrier capsule (Fig. 8b1), P(NVPc) promoted the “free-oating” of the liposomal sub-units in the void of the carriercapsule (Fig. 8b2).93 Since P(NVPc) is a cholesterol-containinguncharged block copolymer and contains the cholesterolmoieties in a short block, while the others are copolymers, wespeculated that in the former case the entanglement of theliposome-anchoring polymer with the carrier capsule is mini-mised and, therefore, allows for the detachment of the lipo-somes from the polymer membrane. The cholesterol-modiedpolymers allowed us to assemble capsosomes with up to160 000 liposomes within a 3 mm diameter carrier capsule.249

Further, P(NVPc), PMAc, or PMAoa, allowed for the subsequentassembly of the carrier capsule using the polymer pair P(NVP)and thiol-modied poly(methacrylic acid) PMASH via hydrogenbonding, followed by crosslinking of the thiols in the lm250 andP(NVP) release at physiological conditions upon disruption ofthe hydrogen bonds.251 Their functionality and their potential

(4) shows an identical experiment, but for a liposome mixture using bivalentcoupled chol–DNA. Reprinted with permission from ref. 224. Copyright (2005)American Chemical Society. (b) Site-specific immobilisation of liposomes con-taining GPCRs is shown using ZeptoREADER imaging. Reprinted with permissionfrom ref. 225. (c) (i) Schematic illustration of the preparation of cisplatin, (cis-diammine dichloroplatinum(II); CDDP) liposomes. Adapted with permission fromref. 158. (ii) In vivo CDDP concentration in kidney tissue of normal male ChineseKun Ming (KM) mice, at predetermined time intervals after injection of CDDPloaded liposomes and free CDDP. Reprinted with permission from ref. 158.



Fig. 8 Capsosomes: (a) schematic illustration of the capsosome assembly. A silicaparticle (i) is coated with a cholesterol-functionalised polymer precursor layer (ii),liposomes (iii), and a cholesterol-functionalised polymer capping layer (iv), fol-lowed by subsequent polymer multilayer deposition (v), and removal of the silicatemplate core (vi). Liposomes are adsorbed onto the polymer surface non-cova-lently with cholesterol-modified polymers (iva). Cholesterol is spontaneouslyincorporated into the lipid membrane (ivb). Reprinted with permission from ref.



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for biomedical applications were conrmed by encapsulatingboth hydrophilic52,93,99,249 and hydrophobic248,252 cargo within theliposomal sub-compartments and the demonstration of thepayload activity in cell viability assays248,252 and for encapsulatedenzymatic catalysis.52,93,253 A particularly interesting example is atwo-step catalytic reaction in which the production of asubstrate was employed to subsequently trigger cargo release byco-encapsulating glutathione reductase (GR) within cholesterol-containing liposomes and a disulde-linked polymer–oligo-peptide conjugate within the polymer carrier capsule(Fig. 8ci).253 By incubating these capsosomes with oxidisedglutathione (GSSG) and by increasing the temperature above theTm of the liposomal subunits, the entrapped enzyme wasallowed to interact with the substrate GSSG and converted itinto its reduced product (GSH) which subsequently inducedthe release of the encapsulated uorescent oligopeptides fromthe capsosomes by reducing the disulde bond, as shown by thedecrease in uorescence intensity over time (Fig. 8cii). Thisresult brings capsosomes a step closer to potentially address areal medical problem by continuously generating a potentantioxidant while releasing a small molecule with therapeuticrelevance.

Conclusions and future perspectives

This feature article gives an overview over the current use ofcholesterol in bionanotechnology, including some fundamentalaspects in cholesterol chemistry and polymer chemistry, theapplication of these polymeric materials into self-assembledstructures, and the development of higher order nano-struc-tures, along with their use in biosensing, analytical sciences,drug delivery, and cell mimicry.

In terms of cholesterol chemistry, cholesterol chloroformateis commonly used to conjugate cholesterol. Further, esterica-tion and etherication of the cholesterol’s hydroxyl group arecommon ways to conjugate this molecule. A variety of choles-terol-containing end-functional polymers, homopolymers, andco- and block copolymers have been synthesised and used forthe self-assembly of different micelles, nanoparticles, nano-tubes, nanogels, or polymersomes. An interesting avenue thathas not been investigated is conjugation onto cholesterol inother positions, besides the obvious hydroxyl, and how thiseffects its self-assembly and function. Also, the ne-tuning of

99. (b) Fluorescence microscopy images of capsosomes containing fluorescentlylabelled liposomes using PMAc (1) or PVPc (2) as precursor and capping layers.While capsosomes assembled using PMAc contain the liposomal sub-unitsattached onto the carrier membrane, PVPc promotes the free-floating of theliposomes. Reprinted with permission from ref. 93. (c) (i) Release of encapsulatedoligopeptide KP9 from capsosomes triggered by glutathione reductase (GR).Upon increasing the T above the Tm of the liposomal subunits, the entrapped GRinteracts with the oxidised substrate glutathione (GSSG) and it is converted intoits reduced product (GSH) which, in turn, induces the release of KP9 by reducingthe disulfide bond. (ii) Normalised fluorescence intensity of capsosomes encap-sulating GR within the liposomal subcompartments and fluorescently labelledKP9 upon incubation with GSSG and NADPH as a function of time. A decrease inthe fluorescence intensity indicates a release of KP9. Reprinted with permissionfrom ref. 253.

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the self-assembly process of cholesterol-modied polymers toyield structures with increased complexity would be anotheraspect to consider in the future. Although there are a fewexamples where the self-assembled nanostructures have beenused in vivo, many of them were only examined in vitro using amodel drug, if at all. A future challenge will be to considerspecic medical conditions which require therapeuticcompounds to be delivered and to assess the pharmacokineticsand pharmacodynamics of the cholesterol based deliveryvehicle under biologically relevant conditions. An importantquestion to address would be if the cholesterol-based deliveryvehicles would inherently have an impact on cells or the healthof animals in vivo.

Cholesterol has the ability to self-assemble from multi-component solutions. Many fundamental aspects are beingunderstood and many applications are being thought of. Thefuture challenge here will be the stabilization and post-modi-cation of helical ribbons in order to integrate them in devicesand use them as a tool in nanotechnology e.g. as nano-springs.

Cholesterol incorporated into liposomes also plays animportant role in biomedical applications. Its ability to modu-late the physicochemical properties of lipid bilayer has beenemployed to stabilise liposomes for their application in drugdelivery or analytical science. In the former case, in order tomake more liposome-based formulations commercially avail-able for clinical applications, alternative concepts for stabili-zation and functionalization will have to be conceived. Whilecholesterol will remain an integral building block, cholesterol-modied polymers, instead of (cholesterol-)modied lipids, as away to improve the stability and control over cargo retentionand release, should be considered more. In analytical science,the use of cholesterol has a well-dened task, i.e. to stabilize themembrane. The future challenge in this context remains tosuccessfully move from proof of concepts in research lab tocommercially available products.

Further, cholesterol can be used to anchor other moleculessuch as PEG or DNA to the liposomes for their application inbiosensing or as “stealth” drug carriers. In the latter case, thechallenges are similar to the ones outlined above. One of theaims when using DNA-tagged liposomes in biosensing is toassemble a single-liposome array with the liposomes carryingdifferent membrane proteins, and to nd a means to analysethe protein–analyte interaction in a fast, quantitative andsensitive way. This would contribute considerably to the earlydetection of disease and identication of suitable drugcandidates.

Cholesterol-modied polymers were found to be essential inthe assembly of capsosomes as simple cell mimics. These havebeen well-characterized from a material point of view anddiverse functionality has been demonstrated. In our research,we are convinced that capsosomes have the potential toaddress clinical issues, when the proper route of administra-tion and the suitable cell type to mimic is chosen. However, wehave yet to perform in vivo experiments and to consider actualmedical conditions. These are major future challenges to beconsidered for further development of capsosomes inbiomedicine.

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Overall, cholesterol is a small molecule which has had agreat impact in all the elds presented, and it will remain anindispensable player in various areas of bionanotechnology.

Acknowledgements

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

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