Hosta Rigau L 2013 Ns Cholesterol A A Biological Compound

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

in bionanotechnology
Cite this: Nanoscale, 2013, 5, 89
Leticia Hosta-Rigau,a Yan Zhang,ab Boon M. Teo,a Almar Postmac

Published on 05 November 2012 on http://pubs.rsc.org | doi:10.1039/C2NR32923A
and Brigitte Städler*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
Received 26th September 2012
Accepted 31st October 2012
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
DOI: 10.1039/c2nr32923a
impact ranging from biosensing to drug delivery is outlined. Cholesterol is and will be an indispensable
www.rsc.org/nanoscale player in bionanotechnology, contributing to the progress of this potent field of research.
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Introduction to copy the agella of bacteria4,5 or the use of motor proteins6

have been considered. In the former case, the self-assembly of
Bionanotechnology is aiming to learn from nature’s approach DNA,7,8 peptides,9,10 or lipids11,12 into a variety of nanostructures
to assemble and run “nano-machines” and to mimic these has been demonstrated and their complexity is permanently
concepts by engineering functional nano-devices e.g. making increasing and functionality is implemented. Conjugates with
use of the self-assembly capability of biological building blocks biomolecules e.g. cholesterol, have proven to be indispensable
or imitate nature’s way of motility.1–3 In the latter case, attempts building blocks. Cholesterol, an essential component of
mammalian cells, has been described by Michael S. Brown and
a
iNANO, Aarhus University, Aarhus, Denmark. E-mail: bstadler@inano.au.dk; Tel: +45 Joseph L. Goldstein as “the most highly decorated small mole-
8715 6668 cule in biology”,13 and this statement could probably be
b
State Key Laboratory for Modication of Chemical Fibers and Polymer Materials, extended for its application in bionanotechnology as well.
College of Material Science and Engineering, Donghua University, Shanghai 201620,
Looking back, the term “cholesterol” was introduced in the
People’s Republic of China
c
beginning of the 20th century from the term “cholesterine”,
CSIRO Materials Science and Engineering, Ian Wark Laboratory, Bayview Ave,
Clayton, Victoria 3168, Australia coined by Chevreul in 1816 “Je nommerai cholesterine, de colh,
Leticia Hosta-Rigau received her Yan Zhang is currently a PhD

PhD in Chemistry from the student in the College of Mate-
University of Barcelona, Spain, rial Science and Engineering at
in 2009, under the supervision Donghua University, China.
of Prof. F. Albericio. Her Prior to this, she received her MS
research was focused on the degree in Tianjin Polytechnic
synthesis of peptides with anti- University, China. Currently,
tumor properties by Solid Phase she works as an exchange PhD
Peptide Synthesis. During 2010– student in the group of Assistant
2011 she worked as a post- Prof. Brigitte Städler at iNANO,
doctoral researcher in the group Aarhus University, Denmark.
of Prof. F. Caruso at the Her research involves the
Department of Chemical and assembly of advanced capsules
Biomolecular Engineering at the University of Melbourne, Aus- towards their application in
tralia. Currently she is a post-doctoral research fellow at iNANO, drug delivery.
Aarhus University, Denmark, where she received a Marie Curie
individual fellowship. Her main interest is the development of
articial cells towards a functional biomedical platform with
potential in enzyme-therapy.
This journal is ª The Royal Society of Chemistry 2013 Nanoscale, 2013, 5, 89–109 | 89
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Nanoscale Feature Article
bile, et 23r3o2, solide, la substance cristallisée des calculs biliaires by X-ray studies,19 allowed the established formula to be arrived
humains” (I will call cholesterin of colh, bile, and 23r3o2, solid, at as described in reviews by Windaus and Neukirchen.20,21
crystalline substance of human gallstones).14 However, choles- In nature, cholesterol is heterogeneously distributed in
terol as a substance was already known in the late 1700’s as an cellular membranes, with the highest content found in the
alcohol soluble fraction separated from human gallstones plasma membrane.22 Most nucleated cells synthesise choles-
observed by Poulletier de la Salle. In 1789 De Fourcroy had terol in a regulated process consisting of multiple steps that
prepared a large quantity of this substance, believing that it is mainly take place within the endoplasmic reticulum (ER).23–25
the same as “blanc de baleine” or spermaceti.15 He labeled the Alternatively, cholesterol can be taken up by the cells from
crystalline fraction “adipocire” or fatty wax, a material similar to lipoproteins.26 There are various pathways to traffic cholesterol
that obtained from acid treated grave wax. (Grave was a to the plasma membrane in order to maintain the high
substance formed normally in damp areas of cemeteries from concentration.27 When the cholesterol level reaches a threshold
the decomposition of cadavers.) Chevreul later showed that it level which is regulated by the sphingomyelin content of the
differed from these compounds by treatment with potassium cell, cholesterol is transported to the ER for esterication and
hydroxide and melting point comparisons.16 storage.28
Initial forays into elucidation of the structure of cholesterol The rst link between cholesterol and human health was
showed it to be an alcohol17 in the course of the preparation of discovered in 1843, when Vogel showed that cholesterol was a
esters, and later it was shown to be a secondary alcohol18 major component of arterial plaques,29 and it has since then
through its conversion into the ketone cholesterone. The pres- been intensively investigated.30,31 Cholesterol was consequently
ence of the double bond was established in 1868. Further determined to be widely distributed in the animal kingdom and
structural elucidation through parallel studies on derivatives of its isomeric forms, phytosterol, were shown to frequent the
cholesterol and chemically related bile acids, with verication vegetable kingdom.32 Sterol research in the mid- to late 20th
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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 the
Boon M. Teo received her PhD
regulation of cholesterol synthesis and human physiology.36,37
from The University of Mel-
Cholesterol’s predominant characteristic is its ability to
bourne, Australia in 2010
modulate the physicochemical properties (e.g. uidity23 and
working on ultrasonic polymer
permeability38) of the cell membrane. Due to this, cholesterol
synthesis. Currently, she is
affects a number of cellular processes. For instance, cholesterol
working as a post-doc in Assistant
inuences the behaviour of membrane proteins e.g. too much
Prof. Brigitte Städler’s group at
cholesterol and ordering will slow down the diffusion of
Aarhus University, Denmark. Her
membrane proteins and increase the bending modulus of the
research interests include emul-
membrane.39 Cholesterol also acts as a precursor for the
sion polymerization, anisotropic
synthesis of bile acids40 (which are stored in the gallbladder and
polymer colloids and drug
help to solubilise fats), and is also important for the metabo-
delivery systems.
lism of fat soluble vitamins, including vitamins A, D, E, and K.41
Almar Postma received his PhD Brigitte Städler received her PhD
from The University of New from ETH Zürich, Switzerland,
South Wales, under the supervi- followed by over two years as a
sion of Prof. T. P. Davis and Drs post-doc in the group of Prof. F.
G. Moad & M. O’Shea (2005) in Caruso at the University of
the elds of controlled radical Melbourne, Australia. Since
polymerisation and reactive 2010, she is Assistant Professor
extrusion. From 2005 to 2008 he at iNANO, Aarhus University,
held a postdoctoral fellowship Denmark aer she obtained a
position with Prof. F. Caruso at Sapere Aude Starting Grant from
The University of Melbourne the Danish Council for Inde-
working on both polymer-based pendent Research, Technology
capsular drug delivery vehicles and Production Sciences. Her
and with a startup company (iCeutica) in pharmaceutical nano- research group works on the development and characterization of
particulate reformulations. He re-joined CSIRO as a Research smart nature-inspired drug delivery vehicles and on the estab-
Scientist in 2008. His current research interests lie at the interface lishment of microuidic platforms to address questions in
of polymer design/synthesis and their applications in nano- biomedicine.
biomedicine and electoactive materials.
90 | Nanoscale, 2013, 5, 89–109 This journal is ª The Royal Society of Chemistry 2013
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Feature Article Nanoscale
It is the major precursor for the synthesis of vitamin D and both a polar hydroxyl group on one side and a non-polar tet-
various steroid hormones.42 Recently, cholesterol has also been racylic steroidal ring structure with a branched hydrocarbon tail
found to play an important role in membrane trafficking, sort- on the other (Fig. 1a). Further, cholesterol is comprised of a
ing processes, and cell signalling processes, where researchers rigid planar subunit due to the trans-conguration of the rings
have suggested that it is involved in the formation of lipid ras and a exible iso-octyl side chain ‘tail’. The combination of this
in the plasma membrane, as represented by G protein-coupled rigidity and exibility gives cholesterol the property of orienta-
receptor signalling.39,43,44 tional ordering and display of liquid crystalline behaviour in
The aim of this feature article is to outline the impact that combination with amphiphilic lipid membrane binding
the small biomolecule cholesterol has made in the eld of capabilities.
bionanotechnology (Scheme 1). We are reviewing aspects and
progresses in this area made possible and/or considerably
improved and facilitated due to the presence of cholesterol. In Conjugation of cholesterol
the rst part, we will outline the chemistry of cholesterol, The majority of conjugation strategies used to conjugate
starting with fundamental aspects, followed by cholesterol cholesterol onto molecules, biomolecules, or macromolecules
modications and, nally, the synthesis of cholesterol-con- of interest rely on the hydroxyl group in the 30 position of the
taining polymers will be discussed. The next section will cholesterol ‘A’ ring (Fig. 1b). The most common are esterica-
address the state-of-the-art and challenges involving the self- tion, carbamate, and carbonate bond formation using
assembly of cholesterol and cholesterol-modied polymers into chloroformate derivatives. These strategies allow common
nanostructures. The last part of this feature article will consider carboxylic acids, amines, or hydroxyl functional groups which
the effect of cholesterol on liposomes in bionanotechnology are found on the most biologically important or other relevant
either as a stabiliser in drug delivery and in analytical science, molecules, to be coupled onto cholesterol. Other strategies
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or as an anchoring unit in engineering of biosensors, for the applied to conjugate to cholesterol are illustrated in Scheme 2
decoration of liposomes with polyethylene glycol (PEG), or in and discussed in the next paragraphs.
the assembly of capsosomes. The involvement of cholesterol in Of further interest, Achalkumar et al. have recently reviewed
each of these aspects will be demonstrated by giving selected the chemistry and approaches used to make cholesterol
examples. Overall, we hope to demonstrate that the small conjugate linkers for tethering onto phospholipid bilayers.45
molecule cholesterol contributes signicantly to the eld of A practical challenge with any conjugation strategy used for
bionanotechnology and its many applications. obtaining amphiphiles based on cholesterol is the choice of
solubilising conditions. The challenge is to nd a solvent that
The chemistry of cholesterol will dissolve both the hydrophobic cholesterol, and, at the same
time, the hydrophilic material onto which it is conjugated.
Cholesterol is an amphiphatic molecule, a compound pos- Typical solvent choices range from chloroform, dichloro-
sessing both hydrophilic and lipophilic properties. It contains methane, and dioxane, to tetrahydrofuran (THF). Furthermore,
Fig. 1 (a) A structural depiction of a cholesterol molecule highlighting the four

domains of functional importance ((A) important for polarity and hydrogen
bonding, (B) sterol rings, its conformation is important as it provides a rigid planar
skeleton, (C) controls the conformation of the side chain, and (D) branched
Scheme 1 Schematic illustration of the applications of cholesterol (a small aliphatic side chain) (left) to function in membranes as a flat elongated
biomolecule found in biological cell membranes (top left)) in the field of biona- compound. The structure presumed to form in cellular membranes (right).
notechnology: self-assembly of cholesterol-containing polymers into nano- Reprinted with permission from ref. 37. Copyright (2011) American Chemical
structures e.g. polymersomes (top right), cholesterol as a constituent of liposomes Society. (b) Cholesterol molecule showing the tetracyclic steroid frame (A–D) with
(bottom right), and cholesterol as an anchoring unit for liposomes e.g. for cap- numbering of carbon atoms of the sterol nucleus and side chain based on the
sosome assembly (bottom left). conventional numbering system.17,254
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methacryloyl chloride,51–53 allow appropriate vinyl esters to be

synthesised in high yields. Further, the acid chloride can be
obtained via reaction of the appropriate carboxylic acid with
thionyl 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 esterication
using N,N0 -dicyclohexylcarbodiimide and DMAP, has been used
successfully to react a carboxylic acid with the alcohol of
cholesterol (Fig. 2a).57,58 Ring opening of succinic anhydride
with cholesterol can also be used to form a cholesterol ester
with a terminal carboxylic acid which can then be used for
further chemical attachments.59
Etherication
Ether formation from the hydroxyl group is a common strategy
for the attachment of PEG and sugars to cholesterol. For
example, cholesterol functionalised monomers containing a
diethyleneglycol60 spacer and tetraethylene glycol10 spacer,
attached via an ether bond, have been synthesised. Williamson
ether synthesis is also a popular synthetic approach which can
Scheme 2 Schematic illustration of synthetic opportunities and conjugation
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strategies to the cholesterol alcohol. Clockwise from the top: etherification

through the trichloroacetimidate approach and via Williamson’s ether synthesis,
dioxaphospholane cyclic phosphate functionalisation, carbamate and carbonate
conjugation through a chloroformate, and esterification.
derivatives based on cholesterol oen have a strong tendency to

self-assemble. This oen complicates chemistry isolation
procedures such as water washes during solvent extraction
steps. Nonetheless, chemical synthetic procedures for coupling
cholesterol are well represented in the literature to indicate that
these issues can be overcome.
Chloroformate
A common strategy for cholesterol conjugation is the use of the
chloroformates. Cholesterol chloroformate, a commercially
available compound, is simply combined with the material to be
conjugated onto, which carries an amine, to give a carbamate
cholesterol derivative. The addition of the components is
usually performed under cooled anhydrous conditions and in
the presence of a catalytic amount of a base like triethylamine or
4-dimethylaminopyridine (DMAP). This strategy has been used
for the coupling of cholesterol onto, for example, the primary
amines of 30 -O-(6-aminohexyl)-uridine46 or polyethylenimine
(PEI).47 Also commonly applied, alcohols can be coupled to
cholesterol 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 short
spacer between the cholesterol and the vinyl group.48
Esterication
The hydroxyl group lends itself well for esterication reactions
Fig. 2 Schematic examples of cholesteryl-functional polymers: (a) bis-cholesteryl
and, as such, several approaches have been used. Acylation of end-functional P(HPMA),57 (b) ABA triblock copolymer (PNAM-b-PChA-b-
the hydroxyl moiety of cholesterol with commercially available, PNAM),94 and (c) poly(amidoamine)-co-(amidoamine disulfide cholesteryl)
functional acid chlorides, such as acryloyl chloride49,50 and copolymer.102
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be carried out by reaction of the alkoxide of the alcohol with an cholesteryl-functional initiator. An example is the use of a cho-
alkylating agent carrying the ‘R’ group. Alternatively, to lesteryl radical initiator in conjunction with a cholesteryl-func-
combine two alcohols together, one of them can be initially tional vinyl monomer. In this case 4,4-azobis(4-cyano-1-
tosylated (using p-toluene sulfonyl chloride) and allowed to cholesteryl) pentanoate azo initiator was used to initiate the
react with the other alcohol to form the corresponding ether. polymerisation of sodium 2-(acrylamido)-2-methylpropanesulfo-
This was successfully carried out for the synthesis of a norbor- nate to give a cholesteryl end-capped and linear poly(sodium 2-
nene based monomer61 and a vinyl monomer containing an (acrylamido)-2-methylpropanesulfonate).54 In another report,
aliphatic spacer.62 poly[N-(2-hydroxypropyl)methacrylamide]s (PHPMAs) with a
A glycolipid library based on acylated cholesteryl-b-galacto- terminal cholesteryl moiety either at one or both ends of the
sides was synthesised for the elucidation and construction of a polymer chains have been synthesised (PHPMA-Chol and
vaccine against Lyme disease. Here, the glycosylation of PHPMA-2-Chol). These were prepared by the radical polymerisa-
cholesterol with galactose, essentially an ether bond formation, tion of N-(2-hydroxypropyl)methacrylamide, initiated with 4,40 -
was achieved using a trichloroacetimidate approach.63 Several azobis-[(3-cholesteryl) 4-cyanopentanoate] in the presence of
3-cholesteryl 6-(glycosylthio)hexyl ether glycolipids were also 2-mercaptoethanol and thiocholesterol chain-transfer reagents,
prepared via thiol alkylation using 3-cholesteryl 6-iodohexyl respectively.73
ether. The latter was formed from cholesteryl p-toluenesulfo- A cholesteryl functional reversible addition fragmentation
nate via three steps.64 chain transfer (RAFT)74–78 agent with a single cholesteryl func-
tionality placed in the RAFT re-initiating the ‘R’ group was used
Miscellaneous chemistry for the synthesis of u-cholesteryl functional poly(polyethylene
An example of the cyclic phosphate functionalisation of glycol) acrylate.58 A bis-cholesteryl functional ‘R’ group RAFT
cholesterol was published by Iwasaki and Akiyoshi as a cho- agent was used to place two cholesteryl u-end groups onto
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lesteryl-functional monomer strategy for the synthesis of poly(N,N-dimethyl acrylamide) (PDMA), PHPMA and copoly-
biodegradable and biocompatible polyphosphates.65 The mers thereof.57
synthesis of this monomer was achieved by the addition of Atom transfer radical polymerisation (ATRP)79–81 has also
cholesterol to a cooled solution of 2-chloro-2-oxo-1,3,2-dioxa- been used as a tool to initiate polymerisations with a choles-
phospholane in ether. However, only a 33% yield was achieved teryl-functional initiator.82 Xu et al. applied this technique to
aer recrystallisation. synthesise a biomimetic amphiphile from 2-(methacryloyloxy)
ethyl phosphorylcholine from 10-cholesteryloxydecanyl-2-bro-
Monomers and polymers moisobutyrate.83,84 Similarly, cholesteryl-2-bromoisobutyrate
was used to initiate various oligo(ethylene glycol) (meth)acry-
The conjugation techniques previously mentioned are lates to produce copolymers with u-bromine end-groups. These
commonly applied to the synthesis of cholesterol-based (cho- bromine end-groups were later transformed to reactive azides
lesteryl) monomers for the synthesis of polymers. Esterica- by nucleophilic substitution with sodium azide and, once
tions and carbamate formation are typically used to add the assembled into micelles, allowed the azide–alkyne cycloaddi-
appropriate polymerisable or polymer initiating group to tion “click” reaction to occur in the corona of the micelle.56
cholesterol. These can then be used to create a diversity of
polymer architectures such as end-functional polymers,
Post-functionalisation of end-groups
homopolymers, copolymers and block copolymers.
The exibility of RAFT agents allows for a symmetrical design of
Polymer end-functionalisation polymer end-group functionality via the RAFT agent. This can
be achieved by using a RAFT agent that has a central bis-func-
Ring opening polymerisation (ROP)66 is one approach that has
tional radical leaving/reinitiating the ‘R’ group and two thio-
been used for adding efficiently cholesteryl end-functionality to
carbonylthio ‘Z’ groups on either side. Post-functionalisation
polymers. The polymerisation is simply initiated by an alcohol, in
can then be carried out through the aminolysis of the thio-
this case cholesterol, which conveniently ring opens the mono-
carbonylthio end-groups which transform them into thiols.
mers without the use of a catalyst. This approach has been used
Such an approach has been used to react end group thiols with
to synthesise polyesters, polylactide (PLA), polyglycolide and their
3-cholesteryl 6-iodohexyl ether via nucleophilic reaction to
copolymers, poly(lactide-co-glycolide) (PLGA)67 and, with the
make a,u-di(cholest-5-en-3b-yl-6-oxyhexylthio) poly(N-isopropyl
addition of a catalyst Sn(Oct)2, 3-caprolactone, which ring opens
acrylamide) P(NiPAM).85 A pyridyldisulde poly(oligo (ethyl-
to give poly(3-caprolactone).68 Polycarbonates based on tri-
eneglycol) acrylate) RAFT polymer with a pre-attached bovine
methylene carbonate69 or 2,2-dimethyltrimethylene carbonate70
serum albumin (BSA), was functionalised with a cholesteryl
were also easily synthesised without the use of a catalyst.
group through disulde exchange with thiocholesterol.86
Klok et al. used ROP to great effect to synthesise L-lactic acid
oligomer linkers initiated by cholesterol and terminated by
cholesterol, or bioactive drugs, uorescent markers,71 and Homopolymers
generation 1, 2 and 3 L-lysine dendrons.72 One of the ways of introducing several cholesteryl functionalities
End-functionalisation of a radical polymer with a along the length of a polymer chain is by homopolymerising a
cholesteryl moiety can be performed simply by the use of a cholesteryl-functional monomer. Ruthenium-catalysed ring
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opening metathesis polymerisation (ROMP)87,88 of a monomer ROMP can also be successfully used to synthesise well
functionalised with both norbornyl and cholesteryl moieties has controlled block copolymers containing a cholesterol rich
been utilised to make pendant cholesteryl-functional homopoly- section. Here, a norborene functional cholesterol monomer is
mers with a variety of spacers present between the backbone and polymerised to form the second block from a poly(ethylene
the cholesterol.55 oxide) brush block. These block copolymers are assembled into
A technique that has been used more prolically for intro- nanoparticles for the delivery of doxorubicin and indometh-
ducing cholesteryl-functionality along a polymer chain is the acin, through acid labile carbamate linkers.61
radical polymerisation of cholesteryl-functionalised acrylate or
methacrylate monomers to give pendant cholesteryl function-
ality to polymers. This was looked at as early as the late 60’s and Random or statistical copolymers
70’s by Hardy et al.89,90 and De Visser et al.49,50 More recently, Copolymerisation is a convenient method for the introduction
cholesteryl polymerisation has seen resurgence when paired of cholesteryl pendants into the polymer chain at various mole
with controlled radical techniques like ATRP and RAFT. For percentages. Some examples include the free radical copoly-
example, a homopolymer based on cholesteryl acrylate (CA) merisation of acrylic acid (AA) with 5-acryloyloxypentyl choles-
using RAFT was synthesised and block extended with styrene.91 terate (Ch5A) to give P(Ch5A-co-AA),96 NiPAM with CA to give
P(NiPAM-co-CA),97 HPMA terpolymerised with 6-meth-
acrylamido hexanoyl hydrazine and cholest-5en-3b-yl 6-meth-
Block copolymers acrylamido hexanoate,98 and our own work via RAFT mediated
Zhang et al. designed cholesterol monomers with a hydrophilic copolymerisation to give P(MAA-co-CMA) with a 7 mol%
tetra(ethylene glycol) (TEG) spacer inserted between the meso- cholesterol content.52,99 Researchers at the Australian Centre for
gen and the acrylate (or methacrylate) polymerisable group in Nanomedicine have also recently looked at the copolymerisa-
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order to increase the water solubility of the monomer in their tion of CMA with MAA.100 They varied the CMA content from
dispersion polymerisation system.10 From the poly(methacrylic 2 mol% to 8 mol% and together with dimethylamino ethyl
acid (MAA)-co-PEG methacrylate (PEGMA)) copolymer macro- methacrylates53 and RAFT mediated control, they were able to
RAFT agent, they synthesised the block copolymer P(MAA-co- achieve a CMA content ranging from 2 mol% to 20 mol% whilst
PEGMA)-b-P(Chol-TEGMA) under dispersion polymerisation keeping their polymer molecular weights around 20–24 kDa
conditions, which allowed the formation of a block copolymer with low molecular weight dispersities throughout. The
via self-assembly in minimal steps. In a different report, Liu amphiphilic copolymers showed pH-induced phase transitions
et al. used a diethyleneglycol spacer between the cholesterol and in aqueous media and are envisioned to be useful for
the acrylate and made blocks with (benzyl protected) ascorbate condensing and transporting siRNA due to their lipid
sections.92 In our own research, we designed an amphiphilic membrane destabilizing properties.
block copolymer poly(N-vinyl pyrrolidone) P(NVP)-b-P(CA) An example of a polyphosphate terpolymer containing
which was obtained by polymerising a short CA oligomer pendant cholesteryl functionality, pendant ATRP initiating
section of, on average, 3 units from a P(NVP) macroRAFT groups as well as 2-isopropyl-2-oxo-1,3,2-dioxaphospholane units
agent.93 This macroRAFT agent was obtained via the controlled were synthesised as a biodegradable gra terpolymer, a novel
polymerisation of NVP with a xanthate RAFT agent. Alterna- amphiphilic biomaterial which can function as a nanocarrier.65
tively, diblock copolymers have also been designed by growing Alternatively, post-functionalisation of polymers to introduce
the second block from a cholesteryl methacrylate (CMA) mac- cholesterol has been performed on polymers containing amine or
roRAFT agent with a trimethylsilyl protected hydroxyethyl acid reactive groups, PEI,47 poly(L-lysine) (PLL)52 and sodium
methacrylate (HEMA-TMS), giving P(CMA-b-HEMA-TMS). This alginate,101 giving random copolymers. An interesting example of
block copolymer was further deprotected by the addition of post-functionalisation is the Michael addition polymer poly-
concentrated HCl in a THF solution resulting in the clean (amidoamine) (PAA) (Fig. 2c).102 The nanoparticulate polymer
product copolymer P(CMA-b-HEMA).51 Curiously, the authors network can be obtained using cystamine as a cross-linking agent
reported problems with controlling of the polymerisation of the which can be made to react with 2,20 -dithiodipyridine turning
unprotected HEMA. Additionally, they initially observed diffi- them into linear PAAs with dithiopyridyl side groups that are able
culties polymerising CMA under ATRP conditions with methyl to undergo a thiol exchange reaction with the commercially
2-bromopropionate as the initiator and copper(I) chloride as available thiocholesterol. The resultant products are examples of
bipyridine as the catalyst–ligand system at 90 C. Recently, we amphiphilic PAA–cholesterol conjugates in which lipophilic
have looked at the synthesis of an ABA triblock copolymer in two cholesterol moieties are linked to the hydrophilic PAA chain by
steps from a bis ‘Z’ functional RAFT agent to form a CA mac- disulde bonds. These are relatively stable in blood but are
roRAFT agent, from which two N-acryloyl morpholine (NAM) cleavable under the reductive conditions within cells.
arms can be grown (Fig. 2b).94 We designed the NAM : CA block There are many more examples of cholesterol containing
ratio to be close to 2.5 : 1 to direct the spatial self-organisation polymers, mostly with a focus on optical, liquid crystal and
of the polymer into polymersomes. A bis ‘R’ RAFT agent for the some general bioapplications; however, these are beyond the
design of an ABA triblock with the cholesteryl as the ‘B’ block, scope of this review. The reader is directed to reviews by Zhou
for use in liquid crystalline block copolymers, has also been et al. and Yusa for a more exhaustive list and description of
looked at by Shibaev et al.95 these polymers.103,104
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Self-assembly of cholesterol
As previously mentioned, cholesterol is structurally composed
of different sections which play a role in its interesting self-
assembly behaviour in a variety of multi-component
solutions.105–108
Helical ribbons were rst discovered in human gallbladder
bile, and it was found that these structures form as a precursor
of gallstones upon dilution of bile.107 Konikoff et al. conrmed
that cholesterol is the major constituent of these helical ribbons
by various experimental techniques including density gradient
centrifugation or X-ray diffraction. They also found that the
helical ribbons themselves are precursors of stable cholesterol

monohydrate crystals.106,107 Chung et al. used several model bile
systems which consisted of a mixture of three types of chiral
molecules in water: a bile salt, a phosphatidylcholine, and
cholesterol. They demonstrated that helical ribbons formed
with 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 helical
ribbons with these two distinct pitch angles was not unique to
model bile, but was a general phenomenon for a variety of four-
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component systems composed of a bile salt or surfactants, a

phosphatidylcholine or fatty acids, a steroid analogue of Fig. 3 (a) Sequence and relative stability of metastable intermediates plotted as
a functions of time after supersaturation of bile. Reproduced with permission
cholesterol, and water.109 It was found that in undiluted solu-
from ref. 105. Copyright (1994) National Academy of Sciences, USA. (b) Images of
tions containing cholesterol, surfactants, and fatty acids or a helical cholesterol ribbon. Phase images for angle of 0 illumination (i) and for
phospholipids, cholesterol is solubilised by micelles, i.e. aperture synthesis (ii). Reproduced with permission from ref. 113.
cholesterol was contained in the core. These solutions contain
both the free surfactants and the surfactants contained in
micelles in equilibrium. Upon dilution, the equilibrium shis enables the determination of the spring constant of any helices
from primarily micelles to primarily free surfactants. The solely in terms of its observable geometrical dimensions.111
micelles break up and release the cholesterol, causing the Choi et al. used high-speed synthetic aperture microscopy to
solution to become supersaturated with cholesterol. Subse- image helical cholesterol ribbons in surfactant mixtures, as well
quent dilution leads to the formation of large cholesterol as to measure the thickness of a nanoscale cholesterol helical
monohydrate crystals. However, the exact structures that are ribbon (Fig. 3b) which was determined to be 68 nm.112,113 These
formed and the sequence in which they form depend on the fundamental ndings provide the foundation towards applica-
composition of the solution and the concentration of its tions of the helical ribbons.
components as shown in Fig. 3a.105
In an attempt to assess the properties of helical ribbons, Self-assembly of cholesterol-modified
Starostin and coworkers studied the non-monotonic force-
polymers into nanostructures
extension properties and predicted hysteresis behaviour for low-
pitch ribbons of arbitrary material properties using a new Due to its highly hydrophobic sterol skeleton, good biode-
model for inextensible elastic strips. It explained the rst-order gradability and biocompatibility, hydrophobic cholesterol is a
transition between two different helical states as experimentally good choice for the amphiphilic modication of many water-
observed. Furthermore, they revealed a new uncoiling scenario soluble polymers. Self-assembly of amphiphilic cholesterol-
in which a ribbon of very low pitch shears under tension and modied polymers has been a subject of great interest for the
successively releases a sequence of almost planar loops. They past few decades.71,73,101,114–116 Cholesterol units attached onto
considered their results to be relevant for nanoscale devices macromolecules as pendant(s) or as end-group(s) can induce
such as force probes.110 Also, helical ribbons were thought to formation of structures of well-dened morphology in water as
have potential serving as mesoscopic springs to measure or to a result of specic interactions among the cholesterol moie-
exert forces on nanoscale biological objects. The spring ties.103,104 Though cholesterol has been used to modify many
constants of these helices depend on their submicroscopic kinds of polymers,84,102,117–120 herein we will focus on cholesterol-
thickness. Using quantitative phase microscopy, Khaykovich modied polymers and their self-assembly behaviour with
et al. discovered a quadratic relationship between the radius R potential biomedical applications, e.g. drug delivery.
and the thickness t of helical ribbons that form spontaneously Cholesterol-modied polysaccharides have attracted the
in multicomponent cholesterol–surfactant mixtures. The interest of many researchers. Akiyoshi et al. prepared choles-
quadratic relationship (R f t2) between radius and thickness is teryl-bearing mannans (CHM) and cholesteryl pullulan (CHP).
a consequence of the crystal structure of the ribbons and CHM and CHP can self-organise into nanoparticles and gels in
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semi-dilute solution due to the hydrophobic interaction of the

cholesterol moieties.121 Cholesterol-modied 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 a
model drug. CyA was physically entrapped in the nanoparticles
during the dialysis process, and the drug-loading was on the
order of 6.2 wt% of the CS-CH self-aggregated nanoparticles.122
Yu et al. investigated cholesterol-modied glycol chitosan
(CHGC), and used the CHGC self-aggregated nanoparticles for
doxorubicin loading and delivery.123 They found that doxoru-
bicin release from CHGC nanoparticles was dependent on the
pH, with faster release at lower pH. This aspect was considered
important, because the drug would be more efficiently released
in the tumour tissue than in circulation. They reported that the
pharmacokinetic parameters of doxorubicin changed in mice
and more efficient tumor growth inhibition for doxorubicin-
loaded CHGC compared to free doxorubicin was reported,
making these nanoparticles promising carriers for insoluble
anticancer drugs in cancer therapy. The Zhang group published
a series of reports involving CS-CH124–128 and synthesised
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cholesterol-modied O-carboxymethyl chitosan (CCMC),

cholesterol-modied chitosan conjugate with succinyl linkages
(CHCS) and 6-O-cholesterol modied chitosan nanoparticles
(O-CHCS NPs). The self-aggregation behaviour of the modied
chitosan 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 the
cholesterol moieties played an important role in the size and
the surface property of the self-aggregated nanoparticles.
Comparing the morphology and the stability of self-aggregated
nanoparticles between CCMC and CHCS, the negatively charged
carboxymethyl groups are advantageous for the formation of
well-shaped and stable self-aggregated nanoparticles.126 They
Fig. 4 (i) Molecular structure of the diblock copolymer PEG-b-PAChol. (ii) Self-
studied the drug loading and release property of CHCS and assembled structures of PEG45-b-PAChol in dioxane/water: cryo-transmission
CCMC nanoparticles using Epirubicin (EPB) and paclitaxel, electron microscopy (TEM) image of vesicles of PEG45-b-PAChol10 (left) and cryo-
respectively.124,127 The results showed that the release rate TEM images of vesicles of PEG45-b-PAChol16 (right). (iii) Schematic presentation of
decreased with increasing pH of the media. In PBS, the EPB the model for ellipsoidal smectic polymer vesicle (top) and smectic rod-like
micelles (bottom). Reproduced with permission from ref. 135.
release was very slow (24.9% in 48 h), while paclitaxel was
continuously released over 84 h at 37 C. The biodistribution of
paclitaxel-loaded CCMC self-assembled nanoparticles showed
that these nanoparticles seemed to signicantly increase the polymerisation of the PEG blocks) with different hydrophilic/
uptake of paclitaxel in plasma, liver and spleen, but decreased hydrophobic weight ratios could self-assemble into polymer
the uptake in heart and kidney. Further, the interaction aggregates with different morphologies in THF/water and
between BSA and self-aggregated CCMC and O-CHCS NPs with dioxane/water (Fig. 4ii). Adding water to a dilute solution of
different DS of cholesterol moieties was assessed.125,129 The copolymers in dioxane yielded smectic polymer vesicles and/or
addition of O-CHCS NPs led to the decrease of the a-helical nanobers. By using THF instead, solid spherical aggregates were
content of BSA and the increase of the b-strand content. The obtained upon water addition for the PEG45-b-PAChol copol-
unfolding of BSA by a denaturant such as urea was largely ymer. With proper block sizes and the use of appropriate organic
suppressed upon interaction with CCMC self-aggregated co-solvents, these biocompatible cholesterol-based copolymers
nanoparticles. can self-assembly into aggregates with desirable shapes (e.g.
Cholesterol is oen used to modify PEG polymers leading to vesicles or nanobers) (Fig. 4iii), which have potential applica-
different self-assembly behaviour.130–137 Jia et al. synthesised tions in drug delivery and material science.135 Nagahama et al.
amphiphilic liquid crystal block copolymers consisting of a prepared a novel star-shaped copolymer derivative with choles-
cholesterol-based smectic liquid crystalline polymer block terol end groups, cholesterol-substituted 8-arm PEG-b-poly(L-lac-
(PAChol) and a PEG block (Fig. 4i).135 They found that PEG45-b- tide) (8-arm PEG-b-PLA-cholesterol). Based on the specic
PAChol and PEG114-b-PAChol (45 and 114 are the degrees of self-assembly between the cholesterol end groups, the 8-arm
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PEG-b-PLA-cholesterol aqueous solution exhibited instantaneous

temperature-induced gelation at 34 C.131 In addition, PEGylated
poly(N-methyldietheneamine sebacate)-co-((cholesteryl oxocarbo-
nylamido ethyl) methyl bis(ethylene) ammonium bromide)
sebacate) (PEG-P(MDS-co-CES)) were synthesised, which could
self-assemble into stable micelles of small size with a hydro-
phobic core and hydrophilic shell.136,137 Different cholesterol-
graing degrees of the PEG-P(MDS-co-CES) polymer were shown
to have improved stability of micelle/DNA complexes in blood for
systemic in vivo gene delivery. In another report, Manakker et al.
investigated the rheological properties of a self-assembled
hydrogel system composed of b-cyclodextrin (bCD)- and choles-
terol-derived 8-arm star-shaped PEG.133 They found that the 8-arm

polymer-based mixtures yielded tight viscoelastic networks, but
their storage and loss moduli signicantly deviated from those
predicted by the Maxwell model. Rheological analysis also Fig. 5 Schematic representation of a polymersome (i) assembled from an ABA
showed that the hydrogels were thermo-reversible. At low triblock copolymer consisting of poly(N-acryloyl morpholine) and poly(cholesteryl
temperatures, the gels showed viscoelastic behaviour due to slow acrylate) with entrapped cargo in the hydrophobic part of its polymer membrane
(ii), and a cryo-TEM image confirming the assembly of polymersomes (iii).
overall relaxation of the polymer chains. At higher temperatures,
Reproduced with permission from ref. 94.
however, a reduced number of bCD–cholesterol complexes and
concomitant faster chain relaxation processes eventually led to
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liquid-like behaviour.
In summary, based on the hydrophobic interaction, choles-
Cholesterol-modied compounds can also be mixed with
terol-modied polymers can self-assemble into different
others, and self-assembled into different macroscopic vesicles.
micelles, nanoparticles, nanotubes, nanogels, or polymer-
For example, the nucleoside 20 -deoxy-20 -aminouridine was
somes. The size, shape and mechanical properties of these
modied with cholesterol, and the lipophilic nucleoside 20 -N-(2-
assemblies are designed for encapsulation, storage, release and
(cholesteryl)-succinyl)-20-deoxy-20-aminouridine was obtained.
delivery of therapeutics with potential application in biomate-
When mixed with the unsaturated phospholipid dio-
rials and biomedicine.
leoylphosphatidylcholine, microtubes were formed. From
confocal uorescence microscopy images, the tubes appeared
as open-ended cylindrical structures with outer diameters Cholesterol and liposomes
between 2 and 3 mm and lengths between 20 and 40 mm.138 In
The nal part of this feature article reviews the benets of
another report, the dialysis of a dimethyloxide solution of
cholesterol incorporated into liposomes for biomedical appli-
cholesterol-modied dextran (Chol-Dex) and PLA against water
cations. Two different aspects of cholesterol, namely its ability
yielded hollow polymer nanocapsules with a highly stable
to act as stabiliser for lipid membranes and its ability to link
structure and relatively narrow size distribution.139 The hollow
lipid and polymeric structures together, are discussed. Exam-
capsules were thought to be formed by anchoring of most of the
ples of applications in drug delivery and biosensing will be
amphiphilic polysaccharide onto the surface of swelled aggre-
outlined for both aspects.
gates as a result of phase separation of amphiphilic Chol-Dex
from hydrophobic PLA. The capsules obtained have potential
application as drug carriers or as biomembrane models. Cholesterol as a stabiliser
We recently reported the self-assembly of an amphiphilic Liposomes are among the prominent candidates for various
block copolymers containing cholesterol as the hydrophobic biomedical applications in bionanotechnology. Liposomes are
block into polymersomes (Fig. 5).94 We synthesised an ABA spherical vesicles which consist of one or more lipid bilayer
triblock copolymer with the ‘A’ segment consisting of poly(NAM), membrane(s), encapsulating a volume of aqueous medium
a non-ionic hydrophilic polymer with low-fouling properties (Fig. 6a).140 Glycerophospholipids, sphingolipids, and choles-
similar to PEG, and the ‘B’ segment of poly(CA) as the hydro- terol are the predominant components used for their prepara-
phobic part of the triblock copolymer. We demonstrated the tion. Their structural similarity to natural cell membranes,141
stable, non-aggregated assembly of polymersomes with narrow and their ability to mimic the behaviour of biological cell
polydispersity. We used these polymersomes as reservoirs in membranes make liposomes useful in various applications
poly(vinyl alcohol) composite hydrogels and showed a signicant ranging from drug delivery,142,143 cosmetic,144 and food
decrease in viability of adhering myoblast cells when a cytotoxic science145 to agriculture.145 However, control over the lipid
compound was entrapped in the polymersomes, thus conrming bilayer permeability and stability towards solutes or biomole-
the potential of this system in surface-mediated drug delivery, a cules is oen a challenge and can compromise their applica-
biomedical approach which aims to deliver therapeutic tions in the aforementioned elds. This aspect can be regulated
compounds from the surface of an implantable device or a tissue by adjusting the cholesterol content of the lipid bilayer,146,147
culturing scaffold to adhering cells or to the surrounding media. since cholesterol affects the physicochemical properties of the
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states of a lipid bilayer in the absence of cholesterol: the gel

state at lower temperatures and the liquid-crystalline state at
higher temperatures. The individual lipid molecules of a lipid
bilayer have temperature-dependent mobility (uidity). All
lipids have a characteristic temperature in which they undergo a
transition from the gel to the liquid-crystalline phase, which is
known as phase transition temperature (Tm). The phase
behaviour of the lipid bilayer and thus Tm is determined by the
van der Waals (VDW) interactions between adjacent lipid
molecules. The interaction between the acyl chains of the lipids
is mainly governed by two factors: the length of the chain and
the packing of the lipids in the bilayer, i.e. longer tail lipids have
more area to interact, which will, in turn, increase the strength

of the interaction and, consequently, decrease the mobility of
the lipid. The type of acyl chain (degree of saturation) plays an
important role in how the lipids bind in the bilayer, since an
unsaturated double bond can produce a kink in the alkane
chain which will create extra free space within the bilayer, thus
allowing for additional exibility in the adjacent chains.
Therefore, unsaturated lipids have a signicantly lower Tm
compared to saturated lipids. Incorporation of increasing
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amounts of cholesterol into the lipid bilayer induces an

“intermediate” state by increasing and decreasing the uidity of
the hydrocarbon chain below and above Tm, respectively, and
eventually 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 and
prevents lipid crystallisation, while in the liquid-crystalline
Fig. 6 Cholesterol as stabiliser: (a) schematic illustration of the fundamental self-
phase, the hydrocarbon chains of the lipids which interact with
assembly process from individual phospholipid molecules (i) to bilayer membrane
leaflets (ii), followed by transformation into liposomes (iii). A single bilayer consists the rigid, planar rings of the cholesterol, are partly immobi-
of arranged individual lipid molecules with their hydrophobic tails facing each lised. Specically, cholesterol increases the degree of orienta-
other and their hydrophilic head-groups facing toward the internal and external tion and reduces the rate of motion in the liquid-crystalline
aqueous media (iv). Adapted with permission from ref. 140. (b) Schematic phase of the lipid membrane. This leads to a laterally
representation of liposomes with encapsulated enzymes. Substrates diffuse into
condensed membrane with increased packing density and, as a
the void of the liposome through porin channels, react with the enzymes, and the
product is released back into the solution through diffusion. Reproduced with consequence, higher mechanical strength and lower perme-
permission from ref. 141. (c) Schematic representation of a biosensor assay where ability. Nature makes use of this aspect in many ways, but
a DNA capture probe is immobilised on a polyethersulfone membrane and a DNA nanotechnological applications also rely strongly on this effect
reporter probe is coupled to the surface of a liposome. When a specific E. coli as outlined in the subsequent paragraphs.
mRNA is present, a sandwich is formed between the capture probe, the E. coli
mRNA and the reporter probe, capturing the liposome in the capture/detection
zone. The number of liposomes is directly proportional to the amount of E. coli
Drug delivery
mRNA present. Reproduced with permission from ref. 203. (d) Table containing
most common homogeneous complement mediated liposome immunoassays. Liposomes have been considered as drug delivery vehicles due
Reprinted with permission from ref. 205. (ab, antibody; ag, antigen; BSA, bovine to their colloidal size, ease of preparation, controllable surface
serum albumin; CF, carboxyfluorescein; chol., cholesterol; DCP, dicetylphosphate;
and membrane properties, large carrying capacity, and
DMPC, dimyristoylphosphatidylcholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-
phospho-(10 -rac-glycerol); DNP, dinitripheno(y)l(ated); DPPC, dipalmitoylphos- biocompatibility for over 30 years.150 Since the liposomal shell
phatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine; DSPC, dis- can enclose or bind many different classes of substances, lipo-
tearoylphosphatidylcholine; EA, egg albumin; HSA, human serum albumin; PA, somes are successfully employed as therapeutic agents for the
palmitic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SA, stear- delivery of antibacterial,151 antiviral,152 and anticancer drugs,153
ylamine; and SPH, sphingomyeline).
as well as hormones,154 enzymes,155 and nucleotides.153,156
However, two of the major drawbacks of liposomes as drug
carriers are their oen poor stability in blood circulation upon
lipid membrane. For an in-depth discussion, the reader is systemic application and the sometimes suboptimal control
referred to the reviews by Ohvo-Rekilä et al. or Rog et al.148,149 over cargo retention and release.157 In the latter case, drugs have
The amphiphilic cholesterol is arranged in the lipid membrane to be transported to their site of action quantitatively, or the
by orienting the steroid ring parallel to the hydrocarbon chains therapeutic cargo has to be gradually released.146,147
of the lipids (phospholipids or sphingolipids) and the hydroxyl Cholesterol has served to overcome both of the above-
group encountering the aqueous phase. There are two physical mentioned challenges. Since the addition of cholesterol reduces
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the uidity of the lipid bilayer and increases the stability of the coefficient of the diverse drugs. The results showed that, for
liposomal membrane,158 adjusting the cholesterol content of drugs that follow the transcellular passive transport route, the
liposomes has been one of the prime ways to control the liposome column could be used for a preliminary drug
permeability to solutes both in vivo and in vitro. Typically, half- screening, offering an alternative to the high throughput drug
lives of passively encapsulated drugs range from minutes, in egg screening system based on Caco-2 cells used by pharmaceutical
phosphocholine liposomes, to a dozen hours for cholesterol- companies.175,181 In this context, cholesterol has been important
containing liposomes.159 Owing to this increased cargo reten- as a component of the small-intestine brush-border membranes
tion, the rst liposomal formulation of the antitumor drug which are composed of egg phosphatidylcholine, phosphati-
Doxorubicin (Doxil) was approved in 1995 by the Food and dylserine (PS), phosphatidylethanolamine, and cholesterol.182
Drug Administration (FDA) for medical use in oncology.160,161 Liu et al. reported retention data of numerous drugs utilising
Other cholesterol-containing liposomal formulations of drugs, ILC and liposomes made of these components to mimic the
i.e. Daunorubicin (DaunoXome) used for the treatment of intestinal membranes. The aim was to predict drug intestinal
advanced HIV-associated Kaposi’s sarcoma,162 Amphotericin B adsorption, since absorption of orally administered drugs in the
(AmBisome, Amphotec, and Ableet) for the treatment of intestine (i.e. uptake of the drug through the cell membrane) is
fungal infections in febrile neutropenic patients as well as for the rst step of the drug’s action.175,181,183
the treatment of aspergillosis, candidiasis, and cryptococcosis CAPILLARY ELECTROPHORESIS. CE is an analytical technique
infections refractory to Amphotericin B,163,164 and cytarabine that separates ions based on their electrophoretic mobility with
(DepoCyt) for the treatment of lymphomatous meningitis,165 the use of an applied voltage. Liposome capillary electropho-
were FDA approved in 1996 (DaunoXome), 1997 (AmBiso- resis (LCE) is a recent approach that involves the use of lipo-
me), and 1999 (DepoCyt), respectively, and are still on the somes as coating material184,185 or carrier.171,172 The details of the
market.164,166–168 technique are beyond of the scope of this review and for a more
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Liposomal formulations containing cholesterol of different detailed overview, the reader is referred to other reports.171,172
drugs, such as vincristine sulfate169 or cisplatin (Lipoplatin),170 Here, just two examples to illustrate the importance of
have not been FDA approved yet but are on Phase II and Phase cholesterol are discussed. Wiedmer et al. employed 1-palmitoyl-
III clinical trials, respectively. 2-oleyl-sn-glycero-3-phosphocholine (POPC) and POPC/PS lipo-
somes with various amounts of cholesterol as carriers and
steroidal sex hormones as model analytes. The study investi-
Analytical science gated the inuence of cholesterol on the sizes and electropho-
Interest in liposome technologies and applications for analyt- retic mobility of POPC and POPC/PS liposomes with special
ical purposes is constantly growing, and four major areas of focus on the inuence of cholesterol on possible selective
activity can be identied, namely liquid chromatography (LC), interactions between the steroids and the liposomal
capillary electrophoresis (CE), biosensors, and immunoas- membranes. Their results showed that the presence of choles-
says.140 In all four analytical techniques, cholesterol plays an terol slightly increased the liposomal diameter, and signicant
important role in liposome stabilisation. differences in selectivity between the steroids and the liposomal
LIQUID CHROMATOGRAPHY. The understanding of the inter- membranes were achieved through the addition of choles-
action between cell membranes and different molecules (e.g. terol.186 Alternatively, liposomes have been used as coating
drugs, proteins, and peptides) is of great importance in pre- materials in CE in order to mimic biological cell membranes,
dicting the behaviour of the drug in the organism. In vivo since cell membranes are mainly composed of phospholipids
investigations of the fraction of the drug adsorbed in humans and cholesterol, and the interaction between the liposomes and
and animals are time-consuming and difficult to perform. Since the analytes has been studied.185,187 As an example, Hautala and
liposomes structurally closely resemble natural membranes, collaborators showed the coating of silica capillaries with
they are extensively used in medical and pharmaceutical anionic liposomes comprising POPC, bovine brain PS, and
research as models for mimicking the structure and the func- cholesterol, and evaluated the coating effectiveness by exam-
tion of biological cell membranes.171,172 The use of liposomes ining the interactions of steroids with the coated capillaries.
immobilised within the pores of carrier gel beads as a stationary Their results show that the capillaries can be successfully
phase for column chromatography with an aqueous mobile coated with liposomes and those coated capillaries can be used
phase is known as immobilised liposome chromatography to study the interactions between phospholipid bilayers and
(ILC),173 a method that emerged in the 1990s171 and has proven uncharged steroids. Since the steroids are neutral under the
successful in studying solute–membrane interactions.174 In ILC, applied conditions, the separation is based on hydrophobic
variations in the retention volume depend on the extent of interactions with the liposome coating.187
solute–membrane interaction and can be precisely measured.175 BIOSENSORS. Biosensors show promise in many aspects of
Liposomes can be immobilised in these columns by several medical care from drug development over early detection of
ways173,176–178 including avidin–biotin interaction.140,171,179 This diseases to monitoring the progress of a treatment. All these
specic binding pioneered by Yang et al. allowed prolonging the applications require sensitive, rapid, cheap, and reproducible
column lifetime for over a year.175,179,180 Further, these liposome detection and recent advances in nano- and biotechnology as
columns have successfully been used to predict the drug well as chemistry, biochemistry, physics, and biology have made
absorption in vivo by estimating the membrane partitioning outstanding contributions141 for the development of biosensors
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in medicine,188–190 for environmental applications,191 and in the signal amplication. The method is based on the formation of a
food industry.192,193 However, ways to immobilise biological sandwich between a DNA-capture probe immobilised on a poly-
molecules while preserving their activity remains one of the ethersulfonate membrane, the target mRNA sequence, and a
major challenges in the development of advanced biosensors. DNA-reported probe coupled to the surface of a liposome (Fig. 6c).
Encapsulation techniques are among the most favourable When a specic E. coli mRNA is present, a sandwich is formed
approaches to entrap biological molecules while preserving between the DNA capture probe attached onto the poly-
their functionality for an extended period of time. Various ethersulfonate membrane, the E. coli mRNA, and the reporter
approaches have been employed for the encapsulation of bio- probe attached to the liposomes, thus the liposomes being
logical material including polymeric vesicles,194 biomolecule– captured in the capture/detection zone. Since the number of
nanoparticle hybrid systems,195 capsules assembled via the liposomes is directly proportional to the amount of E. coli mRNA,
Layer-by-Layer (LbL) technique,196,197 or single-enzyme nano- when non-specic RNA is present instead of E. coli mRNA, the
particles where each enzyme molecule is surrounded by a liposomes are not captured in the capture/detection zone, and no
porous composite organic/inorganic network.198,199 However, signal is reported in the detection zone.203
liposomes have emerged as lead candidates by providing a IMMUNOASSAYS. Immunoassay formats rely on a directly or
comfortable cage which can preserve the biomolecule activity in indirectly detectable label. The label can be an easily detectable
a three dimensional space, which is particular important for molecule (e.g. radioactive or uorescent) or, alternatively, an
enzymes.141 Fig. 6b shows a representation of a liposome enzyme. In an enzyme linked immunosorbent assay (ELISA) the
encapsulating enzymes. The substrate can diffuse across the enzyme label generates a measurable amount of product which
membrane through channel gates, react with the enzymes, and is (inversely) proportional to the unknown concentration of
the products can be released back in solution.141 In addition, analyte (usually an antigen).205 The technique essentially
encapsulation offers other benecial properties e.g. amplica- requires any ligating reagent which can be immobilised, a
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tion since liposomes concentrate the enzymes in a conned detection reagent that will bind specically, and an enzyme that
space, thus, the enzymes are able to convert a higher amount of will generate a signal that can be quantied. LISA, liposome
substrate molecules into their corresponding products.172 This immunosorbent assay, is the counterpart of ELISA, with the
feature is benecial in a number of biosensor applications such enzymes replaced by liposomes encapsulating a large amount of
as the determination of pH200 and oxygen,201 or the detection of marker and receptor molecules. The sensitivity of the system is
bacteria202,203 and other agents.204 For the creation of liposomes high and the additional advantage is that, by lysing the lipo-
in biosensing applications, cholesterol plays a crucial role in somes, the detectable markers are released immediately, thus
controlling the uidity and, in particular, the permeability of avoiding the ELISA incubation step in which the substrate is
the lipid membrane in order to ensure stable entrapment of converted into the product.172 Similar to an ELISA, the encap-
molecules required for detection.172 sulated marker can be colorimetrically, uorimetrically, chem-
An interesting example of cholesterol-containing liposomes iluminometrically, or electrochemically detected upon lysis of
in this context was reported by McNamara and co-workers, who the immobilised liposomes.205 Additionally, most formats
developed a uorescence-based oxygen nanosensor by encap- applied in enzyme immunoassays can be used in combination
sulating an oxygen-sensitive dye in the internal compartment of with liposomes.205 The stability of the liposomes is of para-
the liposomes.201 A major concern when developing liposomal mount importance for successful LISA performance e.g. in
uorescence-based sensors is leaking of the dye molecules from terms of sensitivity and selectivity. To increase the stability of
the supportive matrix. To this end, McNamara and collabora- the liposome membrane, cholesterol in combination with
tors prepared liposomes with a lipid composition consisting of saturated lipids has been employed.205
dimyristoylphosphatidylcholine, cholesterol, and dihexadecyl Fig. 6d depicts a table with the most commonly used lipo-
phosphate and showed high stability toward dye leaking at somal compositions and the markers used to detect different
room temperature for 8 days, a fact which could be attributed to analytes. As shown in the table, most liposomal compositions
the addition of cholesterol to the lipid membrane. contain cholesterol to equip the lipid bilayer with additional
Since safety of water and food supplies has to be ensured to stability.205 For instance, Kim and collaborators reported the
avoid contamination of infectious agents, the detection of path- creation of a liposome immunoassay for the detection of genta-
ogenic organisms remains a crucial aspect. With the aim to micin using phospholipase C to release the uorescent marker
address this issue, Baeumner et al. developed a nucleic acid based from the liposomes.206,207 Gentamicin, a drug frequently used for
sensor, also known as a genosensor or gene probe, which is a the treatment of serious Gram-negative microbial infections, was
highly sensitive and specic RNA biosensor for the rapid detec- chosen as a model analyte.206 The liposomes were composed of a
tion of viable Escherichia coli (E. coli) as an indicator of pathogenic mixture of DPPC and cholesterol, since it was previously
organisms in water.203 Briey, the mRNA from E. coli was extrac- demonstrated that this composition allows the lysis of the lipo-
ted, puried, amplied, and then quantied with the biosensor. somes by the lowest concentration of phospholipase C.206,207
The biosensor is a membrane-based DNA/RNA hybridisation
system and cholesterol-containing liposomes (a mixture
composed of dipalmitoyl phosphatidyl choline (DPPC), choles- Cholesterol as anchor
terol, dipalmitoyl phosphatidyl glycerol, and dipalmitoyl phos- So far, the aspect of the cholesterol’s ability to affect the
phatidyl ethanolamine) encapsulating sulphorhodamine for membrane properties of the liposomes and the consequences
View Article Online
for different biomedical applications have been discussed. This were created when it was realised that neither mechanical nor
section considers the use of cholesterol for a different purpose, electrostatic stabilisation could improve their performance
namely as an anchor either to immobilise the liposomes to a signicantly enough.157 It has been widely demonstrated that
surface for biosensing applications, or to coat liposomes with the incorporation of glycolipids such as monosialoganglioside
specic molecules. GM1226 into the lipid bilayer or the coating of liposomes with
hydrophilic polymer chains like polysaccharides226 or, prefer-
Biosensors entially, PEG226,227 becomes an efficient stabilisation tool by
providing a steric barrier. The particular efficiency of surface-
Liposomes hold enormous potential for biosensing applica-
attached PEG chains has been explained by the combination of
tions, not only as labels as discussed previously, but also for the
their water solubility and exibility, which sterically stabilises
establishment of a successful platform for high-throughput
the liposomes by preventing them from self-aggregation and
(membrane) protein sensing.208 Immobilised liposomes in an
fusion, as well as from opsonin adsorption, the rst step
array format can act as supports for (membrane) proteins since
towards clearance by the reticuloendothelial system.226–228 PEG

the lipid bilayer provides a favorable environment that allows
coated liposomes remain in the blood for up to 100 longer
protein exibility and movement while avoiding their denatur-
than ordinary liposomes, thus increasing the pharmacological
ation and loss of function. These sensors are of paramount
efficacy of the encapsulated agents,157 and passive targeting to
importance since proteins play a major role in disease devel-
tumors has been reported.227 The reader is referred to recent
opments and 60–70% of the drug targets are (membrane)
reviews229–232 for a more detailed discussion on the topic of
proteins.209
PEGylated liposomes.
Approaches to immobilise intact liposomes include linkages
There are different ways to coat liposomes with PEG such as
such as avidin–biotin,210,211 interaction between antibody and
by graing it to the lipids by covalent bonding.226 Alternatively,
antigen,212,213 disulde bonds,214 or the chemical linkage
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cholesterol or phospholipids have been employed to anchor

between histidines,215 but also the chemisorption of vesicles
PEG to the liposome membrane, and it has been shown that
onto gold lms.214 However, when these types of linkages are
cholesterol-PEG was easily incorporated, making cholesterol
used, the controlled immobilisation of different types of lipo-
the more successful anchor moiety.104,158,226–228,233–235 They spec-
somes to predened spots on the surface is impossible. This
ulated that long anchor units are difficult to insert into the
challenge has been addressed by tethering liposomes to solid
liposomal membranes or that there is a higher affinity between
supports via DNA-directed coupling, an approach that was rst
cholesterol and the constituent lipids.227 PEG-cholesterol has
introduced by Niemeyer and co-workers.216
the added benet that it confers cohesion on lipid bilayers by
Several strategies have been developed for the DNA-labeling
inducing tighter molecular packing and restricting the motions
of intact vesicles by using functionalised lipids such as thiol-
of the hydrocarbon chains, thus improving the cargo reten-
reactive maleimido-lipids217,218 and thiol derivatives of DNA.219
tion.226 Long-circulating liposomes containing cholesterol–PEG
Alternately, several approaches which make use of cholesterol-
have already been successfully used in the cisplatin delivery in
modied DNA for spontaneous anchoring of liposomes to
vitro and in vivo.158 Fig. 7ci depicts a schematic illustration of
(patterned) substrates with immobilized complementary
liposomes containing a mixture of cholesterol-PEG and thio-
strands have been reported (Fig. 7ai).220–223 This non-covalent
lated cholesterol for cisplatin encapsulation. The in vivo
approach is advantageous compared to the previously
cisplatin concentrations in the kidney tissue of Chinese Kun
mentioned concepts since it is largely independent of the lipid
Ming mice aer injection of the liposome formulation and free
composition of the liposomes, does not require chemical
cisplatin were compared (Fig. 7cii). The results showed that
modication of a lipid, has fast coupling rates, and there is no-
PEGylated liposomes could successfully avoid the peak-
need of introducing functionalised lipids. Therefore, by making
concentration in kidney aroused from free cisplatin, reducing
use of a naturally occurring membrane constituent, unwanted
the in vivo acute nephrotoxicity.158
side reactions between the functional groups and other
membrane constituents are eradicated. However, cholesterol-
based anchoring of DNA is relatively weak, i.e. it is reversible. In Capsosomes
order to allow tagging of a type of liposomes with a specic DNA
Sub-compartmentalised systems hold tremendous potential as
sequence, the cholesterol anchor has to be strengthened by
cell mimics, which is an approach that aims to substitute for
using bivalent cholesterol DNA coupling.224 This approach
lost or missing cellular function.236 Articial multi-compart-
allows sorting of red and green liposomes from a mixture
ment systems do not need to possess the entire complexity of
(Fig. 7aii). In a continuation of this approach, ligand binding to
biological cells, but a specic key feature, which will mostly
liposome arrays equipped with G-protein coupled receptors has
depend on the application in mind (e.g. enzyme encapsulation
been demonstrated on the way to develop this platform into a
for enzyme replacement therapy to provide a biological-inspired
heterogeneous, self-sorting biosensing platform (Fig. 7b).225
long term solution for patients with chronic diseases).
Mimicking the hierarchical sub-compartmentalised structure of
PEGylation cells is essential to perform triggered, spatially separated,
One of the main drawbacks of liposomes in drug delivery is encapsulated (enzymatic cascade) reactions.52,237 Among the
their low stability in systemic circulation. Stealth liposomes reported multi-compartment systems (e.g. vesosomes,238
View Article Online
liposomes within a liposome carrier, polymersomes within a

polymersome,239 polymer capsule within a polymer
capsule,240,241 polymer hydrogel capsules containing cubo-
somes242 or polymersomes243 and cellosomes,244 yeast cells
associated with a polymer membrane)245,246 capsosomes, intact
liposomes embedded into a polymer carrier capsule,52 are a
novel, well-characterised system with potential in therapeutic
cell mimicry. Capsosomes, as a dual composition system, can
take advantage of the different properties of the sub-compart-
ments and the carrier capsule. Liposomes, due to the inherently
high impermeability of their lipid membranes, are ideal to
encapsulate small or fragile cargo. The polymeric carriers, due
to their semi-permeable nature, permit the interaction between

the internal and the external environment while providing the
structural integrity and help to overcome some of the limita-
tions of liposomes such as instability under in vivo conditions
and the lack of control over their degradation. Capsosomes are
assembled via the LbL technique,247 by alternatingly depositing
polymer and liposomes onto silica particles, followed by the
assembly of the polymer carrier capsule and core dissolution
(Fig. 8a).
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With the aim of stably anchoring the liposomes into the

polymer membrane, we employed cholesterol-modied poly-
mers, i.e. cholesterol-containing PLL (PLLc),52,99 poly-
(methacrylic acid-co-cholesteryl methacrylate) (PMAc),52,99 and
poly(N-vinyl pyrrolidone-block-cholesteryl acrylate) (P(NVPc)).93
We also showed that PMAc was the more successful polymer to
anchor liposomes than PMA containing oleic acid (PMAoa).248
Interestingly, while PMAc, PMAoa, and PLLc led to the formation
of capsosomes with the liposomal units associated with the
polymer carrier capsule (Fig. 8b1), P(NVPc) promoted the “free-
oating” of the liposomal sub-units in the void of the carrier
capsule (Fig. 8b2).93 Since P(NVPc) is a cholesterol-containing
uncharged block copolymer and contains the cholesterol
moieties in a short block, while the others are copolymers, we
speculated that in the former case the entanglement of the
liposome-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-modied
polymers allowed us to assemble capsosomes with up to
160 000 liposomes within a 3 mm diameter carrier capsule.249
Further, P(NVPc), PMAc, or PMAoa, allowed for the subsequent
assembly of the carrier capsule using the polymer pair P(NVP)
and thiol-modied poly(methacrylic acid) PMASH via hydrogen
bonding, followed by crosslinking of the thiols in the lm250 and
P(NVP) release at physiological conditions upon disruption of
the hydrogen bonds.251 Their functionality and their potential
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 preferential
adsorption of biotin–BSA to Au spots, leaving the surrounding SiO2 substrate
(4) shows an identical experiment, but for a liposome mixture using bivalent
available for spontaneous supported phospholipid bilayer (SPB) formation, fol-
coupled chol–DNA. Reprinted with permission from ref. 224. Copyright (2005)
lowed by the addition of NeutrAvidin, which binds to biotin–BSA-modified Au
American Chemical Society. (b) Site-specific immobilisation of liposomes con-
only. Biotin–DNAB and chol–DNAA specifically binds to NeutrAvidin/biotin–BSA/
taining GPCRs is shown using ZeptoREADER imaging. Reprinted with permission
Au and SPB/SiO2, respectively, followed by the immobilization of liposomes
from ref. 225. (c) (i) Schematic illustration of the preparation of cisplatin, (cis-
tagged with the complementary DNA. (ii) Micrographs illustrating sorting of
diammine dichloroplatinum(II); CDDP) liposomes. Adapted with permission from
liposomes tagged with different DNA. Micrographs (1) and (2) were obtained by
ref. 158. (ii) In vivo CDDP concentration in kidney tissue of normal male Chinese
exposing the DNA-modified substrate to a mixture of red-labelled and green-
Kun Ming (KM) mice, at predetermined time intervals after injection of CDDP
labelled liposomes tagged with different monovalent coupled chol–DNA. (3) and
loaded liposomes and free CDDP. Reprinted with permission from ref. 158.
View Article Online
for biomedical applications were conrmed by encapsulating

both hydrophilic52,93,99,249 and hydrophobic248,252 cargo within the
liposomal sub-compartments and the demonstration of the
payload activity in cell viability assays248,252 and for encapsulated
enzymatic catalysis.52,93,253 A particularly interesting example is a
two-step catalytic reaction in which the production of a
substrate was employed to subsequently trigger cargo release by
co-encapsulating glutathione reductase (GR) within cholesterol-
containing liposomes and a disulde-linked polymer–oligo-
peptide conjugate within the polymer carrier capsule
(Fig. 8ci).253 By incubating these capsosomes with oxidised
glutathione (GSSG) and by increasing the temperature above the
Tm of the liposomal subunits, the entrapped enzyme was

allowed to interact with the substrate GSSG and converted it
into its reduced product (GSH) which subsequently induced
the release of the encapsulated uorescent oligopeptides from
the capsosomes by reducing the disulde bond, as shown by the
decrease in uorescence intensity over time (Fig. 8cii). This
result brings capsosomes a step closer to potentially address a
real medical problem by continuously generating a potent
antioxidant while releasing a small molecule with therapeutic
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relevance.
Conclusions and future perspectives

This feature article gives an overview over the current use of
cholesterol in bionanotechnology, including some fundamental
aspects in cholesterol chemistry and polymer chemistry, the
application of these polymeric materials into self-assembled
structures, 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 chloroformate
is commonly used to conjugate cholesterol. Further, esterica-
tion and etherication of the cholesterol’s hydroxyl group are
common ways to conjugate this molecule. A variety of choles-
terol-containing end-functional polymers, homopolymers, and
co- and block copolymers have been synthesised and used for
the self-assembly of different micelles, nanoparticles, nano-
tubes, nanogels, or polymersomes. An interesting avenue that
has not been investigated is conjugation onto cholesterol in
other positions, besides the obvious hydroxyl, and how this
effects its self-assembly and function. Also, the ne-tuning of
99. (b) Fluorescence microscopy images of capsosomes containing fluorescently

labelled liposomes using PMAc (1) or PVPc (2) as precursor and capping layers.
While capsosomes assembled using PMAc contain the liposomal sub-units
attached onto the carrier membrane, PVPc promotes the free-floating of the
liposomes. Reprinted with permission from ref. 93. (c) (i) Release of encapsulated
oligopeptide KP9 from capsosomes triggered by glutathione reductase (GR).
Upon increasing the T above the Tm of the liposomal subunits, the entrapped GR
Fig. 8 Capsosomes: (a) schematic illustration of the capsosome assembly. A silica
interacts with the oxidised substrate glutathione (GSSG) and it is converted into
particle (i) is coated with a cholesterol-functionalised polymer precursor layer (ii),
its reduced product (GSH) which, in turn, induces the release of KP9 by reducing
liposomes (iii), and a cholesterol-functionalised polymer capping layer (iv), fol-
the disulfide bond. (ii) Normalised fluorescence intensity of capsosomes encap-
lowed by subsequent polymer multilayer deposition (v), and removal of the silica
sulating GR within the liposomal subcompartments and fluorescently labelled
template core (vi). Liposomes are adsorbed onto the polymer surface non-cova-
KP9 upon incubation with GSSG and NADPH as a function of time. A decrease in
lently with cholesterol-modified polymers (iva). Cholesterol is spontaneously
the fluorescence intensity indicates a release of KP9. Reprinted with permission
incorporated into the lipid membrane (ivb). Reprinted with permission from ref.
from ref. 253.
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the self-assembly process of cholesterol-modied polymers to Overall, cholesterol is a small molecule which has had a
yield structures with increased complexity would be another great impact in all the elds presented, and it will remain an
aspect to consider in the future. Although there are a few indispensable player in various areas of bionanotechnology.
examples where the self-assembled nanostructures have been
used in vivo, many of them were only examined in vitro using a Acknowledgements
model drug, if at all. A future challenge will be to consider
specic medical conditions which require therapeutic This work was supported by a Sapere Aude Starting Grant from
compounds to be delivered and to assess the pharmacokinetics the Danish Council for Independent Research, Technology and
and pharmacodynamics of the cholesterol based delivery Production Sciences, Denmark.
vehicle under biologically relevant conditions. An important
question to address would be if the cholesterol-based delivery Notes and references
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Cholesterol – a biological compound as a building block in bionanotechnology

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Cholesterol – a biological compound as a building block

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

and Brigitte Städler*a

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

Leticia Hosta-Rigau received her Yan Zhang is currently a PhD

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century progressed to biomimetic chemistry,33 tracer work,34

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Fig. 1 (a) A structural depiction of a cholesterol molecule highlighting the four

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methacryloyl chloride,51–53 allow appropriate vinyl esters to be

strategies to the cholesterol alcohol. Clockwise from the top: etheriﬁcation

derivatives based on cholesterol oen have a strong tendency to

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helical ribbons themselves are precursors of stable cholesterol

component systems composed of a bile salt or surfactants, a

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semi-dilute solution due to the hydrophobic interaction of the

cholesterol-modied O-carboxymethyl chitosan (CCMC),

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PEG-b-PLA-cholesterol aqueous solution exhibited instantaneous

terol-derived 8-arm star-shaped PEG.133 They found that the 8-arm

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states of a lipid bilayer in the absence of cholesterol: the gel

more area to interact, which will, in turn, increase the strength

amounts of cholesterol into the lipid bilayer induces an

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towards clearance by the reticuloendothelial system.226–228 PEG

cholesterol or phospholipids have been employed to anchor

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liposomes within a liposome carrier, polymersomes within a

to their semi-permeable nature, permit the interaction between

With the aim of stably anchoring the liposomes into the

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for biomedical applications were conrmed by encapsulating

Tm of the liposomal subunits, the entrapped enzyme was

Conclusions and future perspectives

99. (b) Fluorescence microscopy images of capsosomes containing ﬂuorescently

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of animals in vivo. 1 D. S. Goodsell, Bionanotechnology: Lessons from Nature,

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35 L. J. Goad and T. Akihisa, Analysis of Sterols, Blackie 3924–3934.

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5358. 166 Relevant homepages, http://www.daunoxome.com/, http://

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