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Self-assembly of designer biosurfactants
Debora Berti,* Costanza Montis and Piero Baglioni*
Received 6th February 2011, Accepted 27th April 2011
DOI: 10.1039/c1sm05197k
This contribution reports on some recent advances in the field of self-assembly of biologically inspired
amphiphiles, focusing on nucleolipids. Due to their important potential for biomedical applications,
the synthesis of this class of compounds can be considered a mature field, but precise relationships
between molecular level details and self-assembly properties still have to be elucidated in detail. This
review will present the most recent progresses in the field, along with the new possibilities opened up by
DNA nanobiotechnology, where the hydrophobic functionalization is meant as a tether for
incorporation in soft matrices, such as planar or free standing lipid bilayers.
Introduction
Self-assembly is the main construction pathway for soft matter
systems.1 At the same time it is the principal strategy that Nature
uses to build-up multifunctional systems, whose efficiency,
specificity and energetics have been refined through the iterations
of evolution. Non-covalent interactions between molecular
repeat units are at the basis of self-assembly. A prominent role in
this field is played by surfactant aggregation, where identical
amphiphilic building blocks associate thanks to the solvophobic
interactions of the hydrophobic tails.2 The most obvious natural
counterparts are cell membranes, whose skeleton is formed by
lipid molecules which have an amphiphilic character.3
However, in the bio-world self-assembly is realized not only
due to hydrophobic effects, but it also often results from
a combination of the previous contribution combined with
structural complementarity and specific biological interactions,
generally termed as molecular recognition.4 The similarities
between many biological systems and amphiphilic assemblies,
and the partial conceptual and methodological superposition
between the two research fields, have naturally led to a biomi-
metic fertilization of the surfactant world, directing the evolution
of conventional surfactant molecular units towards the incor-
poration of bio-relevant functions.5 With few notable exceptions,
the chemical modifications mainly concern the polar head
portion and therefore deal with the insertion of biological water-
soluble functionalities.6
The overall aim of this research field is to provide self-
assemblies with some of the biomimetic interactions that Nature
uses in addition to hydrophobic association; in this framework
these systems can be defined as biomimetic self-assemblies.7 The
CSGI and Department of Chemistry, University of Florence, Via dellaLastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy. E-mail: [email protected]; [email protected]; Fax: +39 055 4573032; Tel: +39 0554573033
7150 | Soft Matter, 2011, 7, 7150–7158
term biosurfactant is not used in this review to define surface-
active molecules synthesized by living cells, but rather represents
a contraction of the definition bio-inspired surfactants, and
clearly identifies amphiphiles of synthetic origin.
The most important and paradigmatic examples concern
peptide amphiphiles8,9 and nucleolipids.6,10 The rapid growth of
the use of nucleotides and aminoacid constituents as building
blocks for novel nanostructured materials has been made
possible by the availability of designer custom sequences offered
by the solid-phase peptide11 and oligonucleotide synthesis
methods.12
For peptide amphiphiles, the choice of the sequence includes
about 20 aminoacids with varying charge and hydrophobicity;
this allows a wide selection both for the polar head sequence and
for the hydrophobic tag. When both amphiphilic portions are
formed by aminoacid constituents, the resulting molecule is
called full-peptide amphiphile,10 to distinguish it from the case
where the apolar tail is an alkyl chain attached to the N-terminus
of the peptide sequence. Several examples and reports of self-
assembly from peptide amphiphiles have appeared in the litera-
ture; the latest advances in this field have been extensively
reviewed by Zhang and co-workers, and we refer the reader
interested in this important and emerging area, to this
contribution.11
According to the definition proposed by Rosemeyer in 2005,12
a nucleolipid is a molecule where a nucleic base, a nucleoside,
a nucleotide or an oligonucleotide (see Fig. 1) is covalently
conjugated to a lipid or more generally to a hydrophobic
molecular unit.
The choice for the nucleic portion is surely more limited (four
different kinds of bases in DNA) than for peptide amphiphiles.
The high fidelity of A–T (A–U in RNA) and G–CWatson–Crick
hydrogen-bonding pairing, provides a unique toolbox, despite
the simplicity of this simple four-letter alphabet.13
A nucleo-amphiphile can be globally neutral if a nucleoside or
a nucleic base, both uncharged (see Fig. 1), is derivatized with
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Fig. 1 Chemical structure of DNA chain with the four nucleobases
encircled and the nucleoside and nucleotide structures highlighted.
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a single or a double hydrophobic chain. In some particular cases,
the insertion of cationic groups in the lipid architecture provides
cationic or zwitterionic nucleolipids. The self-assembly and the
applications of these nucleolipids have been reviewed
recently.6,12,14 In the vast majority of examples, when a nucleotide
or an oligonucleotide is used, the molecular unit has a single or
a multiple negative charge. The latter case is the more relevant in
the quest for biomimetic self-assembly.
Oligonucleotide tags can function as molecular zippers
between molecular units or self-assembled objects, provided the
complementarity of the single strands, in a way that is responsive
to temperature and ionic strength.
The limited nucleic bases synthetic library is enriched by
a relevant number of synthetic base analogues which possess
antiviral or antitumor therapeutic efficacy15,16 and can be
included in the ensemble of nucleic base library accessible to
chemists. This research area is also of importance in the field of
enhanced prodrugs, where the lipophilic tag induces self-
assembly that in turn modulates bioavailability.17–19
As we will see, a host of different applications can be envisaged
for the nanostructured organized assemblies arising from self-
assembly of these biosurfactants, from biomedical formulations
to building block for bionanotechnology, to biomimetic systems.
The level of specificity is high by design, therefore the
formulation of organized assemblies from biosurfactants, both
for fundamental studies and for applicative purposes, requires
This journal is ª The Royal Society of Chemistry 2011
a careful consideration of synthetic and chemical details. As the
level of chemical complexity of the molecular units increases, it is
important to maintain structural and dynamical control over the
formed assemblies. This requires an integrated approach to the
field and, considering the application in biomedical and biomi-
metic fields and the novel emerging possibilities in nanotech-
nology, a truly interdisciplinary research strategy.
The focus of this paper is to examine recent advances in the
field of self-assembly of biologically inspired synthetic
nucleolipids.
This subject has been reviewed in the past years,10,12 but it is
undoubtedly a fast growing area. In the following sections, after
an introduction of the synthetic approaches, we will provide an
outline of only the most recent advances in the field.
Nucleolipid synthesis
Synthetic nucleolipid chemistry can be considered a mature field,
whose development has been promoted by the use of such
molecules as drugs or prodrugs, consisting in cytotoxic nucleic
base analogues functionalized with hydrophobic tags. These
molecules are efficient chemotherapeutics, with improved phar-
macokinetic, pharmacodynamic and toxicological properties
thanks to the enhanced uptake from cells and an increased
therapeutic efficacy (mainly antitumoral), associated to lower
adverse effects.
Examples of natural nucleolipids involved in important biolog-
ical processes aremolecules characterizedby antibiotic activity,20as
Tunicamycin,21 which works as an inhibitor of oligosaccharide
synthesis in eukaryotic cells and has demonstrated therapeutic
properties as antimicrobial, antifungal, antiviral and antitumor
agent12,22 or cytidinediphosphatediacylglycerol coenzyme (CDP-
DAG), a natural nucleolipid present both in mammalian and
prokaryotic cells involved in the biosynthesis of glycer-
ophospholipids and in the formation of some membrane compo-
nents like phosphatidylinosides and cardiolipins.23
The important role played by these molecules in biological
systems has inspired the synthesis and investigation of new
nucleolipids characterized by therapeutic activities, and at the
same time has prompted the development of novel synthetic
routes for the derivatization of nucleic acid functions.
The synthetic routes to achieve hybrid lipid–nucleoside/
nucleotide structures can target different atoms for the covalent
attachment of the lipid moiety as shown in Fig. 2.
50-Ribose or deoxyribose functionalization
The first possibility is to bind the lipid moiety to the 50 carbonatom of the sugar ring to obtain nucleoside alkyl esters or
nucleotide phosphate alkyl esters. Examples of this route are the
MacCoss synthesis of 1-b-D-arabinofuranosylcytosine (ara-C)24
and 1-b-D-arabinofuranosyladenine (ara-A)25,26 lipid derivatives,
obtained through the reaction of the nucleoside-50-mono-
phosphate converted to the respective 50-phosphomorpholidate
with the pyridinium salt of L-a-dipalmitoylphosphatidic acid.
The presence of the lipid chain determined for this nucleolipid an
enhancement of the antitumoral effect with respect to naked ara-
A and ara-C, attributed to the enhanced cell uptake and cata-
bolic stability of the lipid derivatives.
Soft Matter, 2011, 7, 7150–7158 | 7151
Fig. 2 Scheme illustrating the different derivatization strategies in the
synthesis of nucleolipids.
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Ara-C analogues have been also studied by Turcotte et al.,27,28
who synthesized compounds derived by either cytosine-50-diphosphate and the corresponding ara-C nucleotide as well as
by Hong et al.,29 who reported the synthesis of new ara-CDP-
steroids and ara-CMP- and ara-CDP-alkyl esters, as reported in
an exhaustive review by Rosemeyer.12
In 2003 Kim and co-workers30 proposed the synthesis of
thymidine-based nucleolipids obtained by using four different
linking units (urea, amide, carbamate and ester) for the attach-
ment of a lipid chain in 50 position. The properties of these
molecules as organogelators were evaluated: fibrous structures
were obtained in urea-linked gelator/cyclohexane systems, woven
structures in ester-linked gelator/toluene and lamellar structures
in amide-linked gelator/toluene. The differences in the organo-
gelators’ gelation ability was attributed to the formation of
mutually exclusive gel networks caused by intermolecular
hydrogen-bonding interactions.
In 1987 Shuto and co-workers31 proposed an enzymatically-
catalyzed reaction for the formation of hybrid lipid–nucleoside
structures. The synthesis of nucleolipids derives from phos-
phocholines and is based on transphosphatidylation reaction
catalyzed by phospholipase D from Streptomyces sp. AA 586
(PLDP) with different alkanols as acceptors. This synthetic route
is particularly relevant for therapeutic applications, due to the
catalytic activity of phospholipases in vivo that provides a meta-
bolic pathway for the degradation of the drug and a decrease of
possible toxic effects. A two-phase CHCl3/acetate buffer reaction
was used for the synthesis of various 50-(3-sn-phosphatidyl)nucleosides carrying pharmacologically active nucleoside head-
groups, such as 5-fluorouridine and arabinocytidine, from the
corresponding phosphatidylcholine and nucleoside PLDP
substrate.32,33 A modification of this synthetic route was carried
out by Berti et al.34–36 for the synthesis of phosphatidylnucleo-
sides characterized by different chains and polar headgroups.
The self-assembly properties of nucleolipids have been investi-
gated highlighting the role of nucleolipid polar headgroups in the
structure of nucleolipid self-assemblies, which will be illustrated
in the following section.
20–30-Ribose or 30-deoxyribose functionalization
Other reactive positions on nucleosides and nucleotides, useful
for the functionalization of nucleic acids and the formation of
7152 | Soft Matter, 2011, 7, 7150–7158
hybrid lipid–nucleoside/nucleotide structures, are ribose 20 anddeoxyribose 30 hydroxy groups.
Schwendener et al.37 reported the synthesis of 30-O-palmitoyl-
nucleolipids of 5-fluoro-20-deoxyuridine by direct acylation with
the corresponding acylchloride. The lipid derivatives showed
a stronger efficacy as anticancer drugs with respect to the naked
fluorinated deoxyuridine; this was attributed to the incorpora-
tion of 30-O-palmitoyl-nucleolipids within liposomes that
provided a protection from degradation inside living organisms.
Barth�el�emy and co-workers presented the synthesis of phos-
phocholineuridineamphiphiles functionalized with two identical
fatty esters on the 20 and 30 positions.38 Dimirystoyl, dipalmitoyl,
distearoyl, diarachidoyl and dioleoyl derivatives were synthe-
sized and their self-assembly properties in aqueous solvent were
investigated. They formed fibers or bilayer supramolecular
structures and evidenced a thermoreversible transformation
from one structure to the other, depending on the melting
temperature of the amphiphiles. A similar fluorinated nucleo-
side-based amphiphile (20,30-O-di-2H,2H,3H,3H-perfluoro-
undecanoyl-uridine-50-phosphocholine) was synthesized with the
substitution of the 20 and 30 nucleoside ribose positions by fluo-
rocarbon chains and its self-assembly properties were investi-
gated.39 Shimizu and co-workers40 presented an example of
nucleotide bola-amphiphiles with a 30-phosphorylated thymidine
moiety at each end of oligomethylene spacers. The structure of
these nucleotide lipid conjugates is compatible with the structural
prerequisites of a good hydrogelator: the sugar and phospho-
diester moieties play an important role in imparting the addi-
tional hydrophilic functionality; the sugar moiety can provide
multiple chiral centers and the thymidine moiety is connected to
both ends of long oligomethylene chains. The oligonucleotide-
templated self-assembly of the thymidine bola-amphiphile with
a series of oligoadenylic acids was also investigated and the
formation of DNA-like nanofibers was obtained thanks to the
Watson–Crick interaction between adenine and thymine bases.41
Amphiphilic cationic uridine derivatives were synthesized42,43
as possible DNA vectors characterized by hydrogen bonding and
p-stacking capabilities, hence capable of summing up to elec-
trostatic compensation between DNA and the synthetic vector.
Moreover, recently the same authors presented the synthesis of
anionic nucleotide based lipids44 featuring thymidine-30-mono-
phosphate as the nucleotide and 1,2-diacyl-sn-glycerol as the
lipid moiety for in vitro delivery of nucleic acids.
Purinic or pyrimidinic heterocycle functionalization
Few examples in the literature report the direct functionalization
of heterocycles’ nucleobases with a lipid moiety. Purine’s N9 and
pyrimidine’s N1, chemically bound to the sugar moiety in
nucleosides and nucleotides, are possible reactive positions for
the attachment of a lipid chain, an example45 is the synthesis of
an acycloguanosine (Acyclovir) lipid derivative with therapeutic
properties for the treatment of Herpes simplex (Type I). An
acylguanosine nucleolipid, effective in the treatment of the same
disease, has been reported by Welch and co-workers.46 Other N9
purine nucleobases–lipid and N1 pyrimidine nucleobases–lipid
hybrid systems were synthesized by Rosemeyer and co-workers
and investigated in monolayers at the air–water interface.47
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Recently Hocek and co-workers48 presented the synthesis of
bile acid-functionalized deoxyribonucleosides (dN) and deoxy-
ribonucleoside triphosphates (dNTPs) with the attachment of the
bile acid moiety in position 5 of pyrimidine and in position 7 of
the 7-deazaadenine nucleosides, obtained by single step aqueous-
phase Sonogashira cross-coupling reactions. The bile acid
modified dNTPs were successfully incorporated in DNA
notwithstanding the presence of a very bulky and hydrophobic
steroid moiety in position 5 of pyrimidine or position 7 of
7-deazaadenine dNTPs.
Oligonucleotide–lipid conjugates
The possibility of synthetic oligonucleotides to interfere with the
regulation of genetic information in biological systems49–51 has
opened the way to their use in gene therapy, and determined
a growing interest also in the development of oligonucleotide–
lipid conjugates. In analogy with nucleoside lipids, the presence
of a hydrophobic segment attached to the oligonucleotide was
thought to promote its interaction with cell membrane and,
hence, to enhance cell uptake. Moreover, many lipophilic
moieties, like cholesterol, can target lipoproteins: therefore
cholesterol–oligonucleotide conjugates can be recognized by
hepatic cells and internalized by lipoprotein-mediated
endocytosis.52,53
The reader is addressed to the articles by Rosemeyer12 and
Gissot et al.17 for oligonucleotide–lipid conjugates, and to the very
recent review byDefrancq et al.54 for oligonucleotide conjugation
methodologies. From a synthetic point of view, it is possible to
insert the lipid moieties during the solid-phase synthesis of the
oligonucleotide sequence, or to modify oligonucleotides with the
hydrophobic moieties once the solid-phase synthesis is accom-
plished and oligonucleotides have been cleaved from the solid
support anddeprotected.Very few examples of the latter synthetic
route are reported in the literature, and the most common and
feasible approach is the first one. The conjugation of oligonucle-
otides is mostly at 50- or 30-termini because of their easy accessi-
bility. Moreover, due to the elongation of the oligonucleotide
chain in the 30–50 direction during the solid phase synthesis, the
modification ismore easily attached to the 50-end, compared to the
30-end; however, it has been suggested that conjugation through
the 30-end enhances the exonuclease resistance. A synthetic route
to achieve 30-end oligonucleotide conjugation was developed by
Letsinger et al.55 Liebscher and co-workers56 also presented the
synthesis of lipophilic uridines containing a lipid moiety into the
20-position. The importance of these 20-lipid functionalized
nucleosides is related to the possibility of introducing themodified
nucleosides into any position of an oligonucleotide strand. The
uridine derivatives containing one or two lipophilic groups in the
20-position, were studied for their incorporation within lipid
membranes and the effect of the exposure of nucleobases to an
aqueous environment for possible base-pairing.
Recently, Tan and co-workers57 proposed the solid-phase
synthesis of DNA–diacyl-lipid conjugate amphiphiles containing
a first segment of single-stranded DNA, highly hydrophilic,
a second segment of a pyrene molecule used as a fluorescence
reporter and a highly hydrophobic third segment composed of
two C18 hydrocarbon tails. When dispersed in aqueous solution,
such amphiphilic DNA spontaneously self-assembled into three-
This journal is ª The Royal Society of Chemistry 2011
dimensional spherical micelles with a DNA corona and a lipid
core. Moreover, micelles disintegrate when incubated with bio-
logical cells through permeation of the cell membrane by endo-
cytosis, opening perspectives for medical applications of these
systems in gene therapy.
Nucleolipid self-assembly: from fundamentals to bio-relevant applications
The self-assembly of amphiphilic molecules produces aggregates
characterized by a large structural diversity, in terms of size,
shape, and interfacial flexibility. The amphiphilic phase diagram
depends on parameters such as surfactant kind, volume fraction,
temperature, and ionic strength.
The main structural features of self-assemblies are related, in
the simplest approximation, to the relative cross-sectional areas
of the hydrophilic and hydrophobic parts, encoded in the
optimal packing parameter of the amphiphile.58 Subtle structural
variations of molecular details can lead to cascade effects with
dramatic morphological transitions on the mesoscale.
The possibility to integrate nucleic motifs into amphiphilic
molecules and use the know-how of soft matter to engineer nano-
objects with nucleic decoration can ultimately lead to extremely
complex and fascinating structures. As the molecular ‘‘bricks’’
become more and more sophisticated and additional control
parameters are chemically introduced, it becomes central to
assess how these added functionalities affect self-assembly and
respond to variation of external conditions. Simple consider-
ations on the relative steric hindrances of the molecular portions
(i.e. the packing parameter) are usually not sufficient, as polar
heads do not merely interact through excluded volume and
electrostatic repulsions.59,60 Such structural complexity requires
an in-depth structural characterization, necessary to highlight
the contribution from base–base interactions to the overall
properties of the assemblies.
An ideal system to test such fundamental ideas is represented
by very simple nucleolipids, i.e. phospholipid-based anionic
nucleolipids, PLN (phosphatidyl nucleosides),31–36 where one
single RNA nucleoside is enzymatically attached to the polar
head of a lecithin substituting a choline headgroup, as already
mentioned in the previous paragraph. These derivatives have
been studied in detail in the last decade. Both the hydrophobic
moiety and the nucleic headgroup have been systematically
varied to modulate the packing parameter and trigger/tune base–
base interactions at the interface of the self-assembled structures.
For a given lipid derivatization, the microstructure of self-
assemblies depends markedly on the kind of nucleic base due to
interpolar-head attractive interactions, notwithstanding the
negative charge, which would rather predict the prevalence of
unspecific electrostatic repulsion.61–63
An illuminating example is provided by dilauroyl-phospha-
tidyl-N (DLPN), with N being either adenosine or uridine, whose
aggregation properties have been studied in detail,64–71 to better
highlight the contribution of the functional polar head to local
and mesoscopic properties of the aggregates.
Freshly prepared DLPA solutions are mostly composed of
threadlike micelles whose cross-section size is about 5 nm. The
observed time evolution of the aggregate morphology results in
the twisted superstructures reported in Fig. 3 and accompanied by
Soft Matter, 2011, 7, 7150–7158 | 7153
Fig. 4 Illustrative cartoon for a possible polyU arrangement in
a nucleolipoplex. For the sake of clarity polyU is here represented as
a rigid rod. (Reprinted with permission from ref. 81.)
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the appearance of an ultraslow relaxation in quasielastic light
scattering, arisen from a hierarchical aggregation of wormlike
structures identical to DLPU micelles. The higher stacking
constants of the former nucleotide are responsible for the different
self-assembly behavior, which is strictly dependent on the thermal
history of the samples. This behavior has been proven to be
dependent on the thermally-activated redistribution of syn–anti
conformations of adenosine headgroups, which is then collectively
frozen thanks to molecular constraints present in the structure.71
Noticeably, mixed complementary phosphatidyl-nucleoside
self-assemblies (micelles, bilayers, etc.) bearing complementary
bases on the polar heads exhibit the signatures of interfacial
interactions both through stacking and specific hydrogen bonds,
according to a Watson–Crick pattern as in nucleic acids.35,36,72–78
Molecular recognition is triggered by self-assembly and affects
the structural features of the aggregates.
These assemblies provide negatively charged interfaces deco-
rated with nucleic motifs that can complex complementary
nucleic acid strands using, as the driving force, molecular
recognition instead of electrostatic interactions. Globular
micelles composed of 1,2-dioctanoyl-phosphatidyl-adenosine
interact with complementary RNA single strands (ss-poly-
uridylic acid), forming a new hexagonal phase where the nucleic
acids are confined among cylindrical micelles.79 This provides
a proof-of-principle that complexation is possible notwith-
standing the same charge of both partners (i.e., lipid assembly
and nucleic acid). The investigation has also been extended to
zero-curvature assemblies (i.e., nucleolipid membranes) formed
by POPNs.80–82 Nucleolipid bilayers of POPA, when swollen with
aqueous solutions containing single-strand complementary
polynucleotides (polyuridylic acid) show an increase in the
smectic period with respect to binary POPA/water systems and
the appearance, upon annealing, of an additional SAXS peak
ascribable to 1D ordering of the biopolymer between the lamellar
stacks, as sketched in the cartoon reported in Fig. 4.81
None of the observed effects could be detected for identically
charged membranes formed by POPG, thus ruling out any
nonspecific effects due to the presence of biopolymer in the
swelling solution.
The extra-peak position scales in the SAXS spectrum with
biopolymer concentration similar to the scaling of lipoplexes.
Such novel structures, sketched in Fig. 4, have been termed
nucleolipoplexes, in analogy to classical lipoplexes.
Fig. 3 Cryo-TEM pictures of micellar aggregates formed by DLPU and
DLPA(1� 10�2M)Thebar is 100nm(Reprintedwithpermissionfromref. 68.)
7154 | Soft Matter, 2011, 7, 7150–7158
The above outlined approach represents a strategic shift from
a specific to tailored vectors for DNA delivery to modulate gene
function in vitro or in vivo. The use of anionic species such as
nucleotide lipids in formulations can optimize transfection effi-
cacy and offer an alternative to cationic species, which can be, in
some instances, cytotoxic.83 This strategy has been successfully
implemented with a similar anionic nucleolipid, diC16-30-dT, byBarth�el�emy and co-workers, who have demonstrated that
complexes with DNA are formed in the presence of calcium ions
and that the biomolecule is internalized into mammalian HEK
cells with enhanced transfection efficacy with respect to the
natural anionic DPPA and DPPG.44
Recently, Drummond and collaborators84 have reported on
the self-assembly of a novel amphiphile pro-drug 5-deoxy-5-flu-
oro-N4-(cis-9-octadecenyloxycarbonyl) cytidine. Similar to
many other nucleosidic chemotherapeutics, the insertion of
a specific lipid carrier linked to the nucleoside-analogue can
reduce the usually severe side effects, improve pharmacokinetic
behavior and overcome or decrease the development of resis-
tance against the drug, considerably improving the quality of life
for the patients. On the other side, the self-assembly pattern
modulates the bioavailability and the bioactivity profile.
The relevance of this study went, however, beyond these
important applicative aspects, to cover fundamental aspects of
self-assembly, yet to be addressed. The authors have observed for
the first time the sequence of structural intermediates, shown in
Fig. 5, in a temperature induced La to QIID transition, following
the formation of interbilayer contacts, the development of
membrane pores, the evolution into a hexagonal structure that
eventually evolves to a bicontinuous cubic structure. These
observations provide mechanistic insights into the formation of
highly ordered cubic membrane structures, exhibited by many
cellular organelles.
Recently, the synthesis of a novel family of glycosyl-nucleoside
lipids with a ‘‘double click’’ chemistry route was reported and
their characterization in terms of self-assembly properties,
described.85 These amphiphiles spontaneously assemble into
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Fig. 5 Schematic representation of the major transformation stages
involved in the creation of a bicontinuous cubic structure from a planar
lamellar structure. (Reprinted with permission from ref. 84.)
Fig. 6 DNA-programmed lipid assembles to form spherical lamellar
vesicles capable of switching phase to form small spherical micelles via
DNA hybridization (+DNA2) and strand invasion (+DNA3) cycles.
(Reprinted with permission from ref. 87.)
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supramolecular structures, including fibers, vesicles, hydrogels,
and organogels, and are able to induce gelation both in water and
chloroform. The delivery of oligonucleotides into human hepatic
cells has also been reported by the same authors.86
The structure-encoded response to external stimuli is one of the
most interesting prerogatives of nucleolipid self-assemblies. The
design of molecular components for a tailored aggregation
behavior can concern the hydrophilic assembler, as the assembly
modulator, or the polar head. In the case of oligonucleotide-
conjugates, the design of the nucleic sequence represents probably
the most powerful and sensitive tool to control self-assembly.
One recent example concerns a very interesting change in
morphology, exhibited by a double-chained oligonucleotide
lipid.87 The design scheme, reported in Fig. 6, is conceived to
enable a programmable soft, discrete nanoscale morphology,
using DNA pairing to encode the amphiphilic building block
with information.
The derivative assembles spontaneously into bilayered vesi-
cles, but, upon pairing with a complementary oligonucleotide,
evolves into a micellar structure, in view of the increase of the
cross-sectional area of the hydrophilic section. If the pairing
oligonucleotide is designed with a dangling end, the subsequent
addition of a third oligonucleotide of identical contour length
and fully complementary to the second one (strand invasion),
reverts the self-assembly from micelles to vesicles, via strand
disassembly at the micellar surface.
Recently Arbuzova and co-workers have reported that
mixtures of simple synthetic phospholipids, like DOPC, with
a novel lipophilic nucleoside 20-N-(2-(cholesteryl)-succinyl)-20-deoxy-20-aminouridin self-assemble in microtubular morphol-
ogies. These authors observed the formation of open-ended
cylindrical structures with outer diameters between 2 and 3 mm
and lengths between 20 and 40 mm after hydration of mixed dry
lipid films, followed by heating to 70 �C and slow cooling to
room temperature. The composition of the initial lipid films and
the conditions of preparation allow some degree of control over
the final morphology of the tubes.88
Nucleolipids for nanobiotechnology
In this last paragraph we will provide some recent advances of
the use of nucleolipid in nanobiotechnology oriented
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applications. The distinction from the cases treated in the
previous paragraph is somewhat arbitrary, as the boundaries
between these areas are actually blurred.
However, in these cases we will refer invariably to oligonu-
cleotide amphiphiles, where the design is operated on the oligo-
nucleotide portion and enables adsorption and orientation of
DNA chains at fluid surfaces including the air–water and oil–
water interfaces. The resulting assemblies are provided with
chemical information that can be translated into further hierar-
chical aggregation in functional arrays of nanounits.
Non-covalent DNA immobilization strategies can be realized
inserting the desired single-strand sequence bearing a hydro-
phobic anchor into a bilayer membrane; in this case the fluid two-
dimensional membrane allows for in-plane free diffusion and
rotation of lipid-associated molecules, thus promoting the
hybridization process at the surface. Therefore the insertion of
a lipophilic tag into an oligonucleotide sequence is solely inten-
ded as the hydrophobic anchor that provides tethering at a lipid/
water interface. It is then clear that oligonucleotide amphiphiles
are ‘‘functional dopants’’ into fluid matrices, typically bilayers;
they are used in very small quantities and their self-assembly is
generally not a concern, while it should be at least considered as
a competitive event with respect to adsorption at fluid interfaces.
Since their introduction89 cholesteryl-TEG conjugates are
generally considered good anchoring candidates since they show
low tendency to self-aggregation and induce a stabilizing effect
on the lipid bilayer. H€o€ok and Benkoski and Boxer et al. pio-
neered the concept that DNA hybridization can be used to tether
vesicles to planar lipid bilayers, at the same time allowing them to
freely diffuse in a plane parallel to the supported bilayer.90 Such
an approach can be envisioned as a useful method for arraying
Soft Matter, 2011, 7, 7150–7158 | 7155
Fig. 7 Mixing/demixing of DNA_tocopherol*/DNAc2* (a–d; FITC-
labeled) and PNA_C16/DNAc1 (e–h; rhodamine-labeled) hybrids
incorporated in POPC/SSM/Chol GUVs due to temperature increase and
decrease. Below the phase transition temperature, the two lipids
DNA_tocopherol*/DNAc2* (a) and PNA_C16/DNAc1 (e) are located to
different domains (�5 �C). Above the phase transition temperature,
domains disappeared, and both constructs mix (b and f) (�55 �C). After
cooling (�46 �C), new smaller domains are formed (c and g) that merge to
larger domains separating again both types of constructs (d and h).
Bars ¼ 10 mm. (Reprinted with permission from ref. 101.)
Fig. 8 Incorporation of cholesteryl-TEG-functionalized oligonucleo-
tides within lipid membranes covering Layer-by-Layer SiO2 nano-
particles. (Reprinted with permission from ref. 99.)
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integral membrane proteins in vesicles and as a platform for
studying their interactions with other proteins, small molecules,
and/or vesicles. Many collisions between individual tethered
vesicles have been observed with fluorescence microscopy, and
the vesicles show no tendency to stick to each other or fuse, while
docking between vesicles occurs if a second complementary
sequence, introduced through 50 lipophilic oligonucleotides, is
present. If vesicle coupling is accomplished through pairing of
50-lipophilic oligonucleotides with the complementary 30-conju-gate, the anti-parallel hybridization mediates vesicle fusion,
revealed by quick lipid mixing and slow content mixing.91–93 This
system represents a mimic of the complex events leading to
protein-mediated membrane fusion in synaptic vesicles, and, by
changing the length of the oligonucleotide sequence, allows
probing the effects of the distance between bilayers in the
process. The incorporation of cholesteryl-tagged oligonucleo-
tides in phospholipid vesicles has been also studied in solution,
establishing that the oligonucleotide conformation and hybrid-
ization kinetics with the complementary strand in solution
strongly depend on the macromolecular density at the liposomal
surface, and above a critical grafting density connected to the
lipophilic oligonucleotide excluded area, coupling kinetics are
slowed down relative to strand pairing in solution.94
Novel synthetic strategies for ss-DNA carrying lipophilic tags
have been recently presented, which allow for the introduction of
lipophilic groups into any position of the oligonucleotide
sequence applying phosphoramidite methodology.95,96 In the
case of multiple cholesteryl derivatives, the effects of competitive
aggregation in solution on adsorption have been elucidated by
comparing single cholesteryl with multicholesterol derivatives in
vesicles and in supported lipid bilayers.97
The construction of a lipid membrane/pseudohexagonal DNA
hybrid, anchored on the membrane through the same choles-
teryl-functionalization has also been reported. The effects of
grafting density, lipid/DNA ratio, liposome number density and
preparation procedure on the final structure and yield of the
resulting hybrid nanomaterial were demonstrated. This work
was the first example of the realization of nonlinear DNA
constructs onto fluid membranes.98 Remarkably, closely match-
ing results for the thickness of the DNA layer have been obtained
for DNA adsorption on liposomes and on SLB at the same
grafting density (Fig. 8).
Very recently, the possibility of creating Janus-like vesicles
with coexisting lipid domains where amphiphilic DNAmolecules
can preferentially partition has been shown. Lipophilic oligo-
nucleotide partition was obtained using tocopherol and choles-
terol-modified DNA.98–102 Upon heating the lateral domains
merge and reform, by cooling these sticky patches would break
the spherical symmetry of inter-vesicle interactions, opening up
the possibility of directional vesicle adhesion. However, exclusive
partitioning of a conjugate into the Lo phase, shown in Fig. 7,
has not been reported until recently.101 Again, inspiration from
Nature has been the elegant guiding principle to design this latter
nucleolipid. To force partitioning into the liquid-ordered phase,
palmitoylation of a DNA-recognizing PNA (peptide-nucleic
acid) has been accomplished in GUV (Giant Unilamellar Vesi-
cles). Palmitoylation of membrane proteins plays in fact in
Nature a decisive strategy to target them to raft domain. The
lateral domains are thermoreversible, as shown in Fig. 7, and
7156 | Soft Matter, 2011, 7, 7150–7158
partition can be thus thermally cycled. This approach offers
a reversible tool for triggering interactions among the structures
and for the arrangement of reactions and signalling cascades on
biomimetic surfaces. This powerful bio-inspired strategy shows
how it is possible to realize synthetic rafts with structural control,
possessing distinct oligonucleotide functionalization and there-
fore distinct and extremely selective coupling functions, and will
presumably have an important impact both in nano-
biotechnology and as a biomimetic system.
An interesting example of the use of oligonucleotide-decorated
vesicles in the hierarchical assembly of simpler units has been
reported recently. Layer-by-layer (LbL) particles were prepared
by the adsorption of polyelectrolyte layers on silica beads and the
outermost polyelectrolyte layer has been modified via covalent
coupling of dA21 oligonucleotides with an amino-linker.102
The outer polyelectrolyte layer provides a template for vesicle
binding via DNA hybridization, resulting in the coating with
a tunable arrangement of several layers of intact liposomes,
functionalized with thymine-based DNA oligonucleotides,
conjugated with two tocopherol moieties and incorporated into
lipid membranes.
This journal is ª The Royal Society of Chemistry 2011
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The disintegration of distinct vesicle layers can be achieved, for
example, by sequence-specific melting of nucleotide bases.
Further applications that can derive from this concept include
the entrapment of different reagents into different layers and
their regulated release on demand.
Conclusions and future perspectives
In this review we have examined the latest progresses in the field
of self-assembly of designer nucleolipids. Nucleolipids join the
self-assembly properties due to the presence of a hydrophobic
assembler with some biospecific functionalities, as those dis-
played by nucleic acids. These properties enrich both the self-
assembly scenario and the range of applications. Innovative
design principles can be applied both to the hydrophobic tail
architecture and to the structure, length and sequence of the
polar head.
From what is reported, it is clear that the field is truly inter-
disciplinary, as many other areas in soft matter. While novel
synthetic strategies enable more and more sophisticated molec-
ular repeat units to be prepared, the challenge will be the control
over the structural and dynamical properties of the self-assem-
blies, and therefore on their possible use to several new
biotechnological applications.
Acknowledgements
This work has been funded by CSGI and the Italian Ministry of
Research (PRIN 20087K9A2J001 and FIRB RBPR05JH2P
Italnanonet).
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