Postprint of: Chem. Soc. Rev., 2011, 40, 1586-1608
Cyclodextrin -based gene delivery systems
Carmen Ortiz Mellet (a), José M. García Fernández (b) and Juan M. Benito (b)
(a) Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado
553, E-41071 Sevilla, Spain. E-mail: mellet@us.es; Fax: +34 954624960; Tel: +34 954559806
(b) Instituto de Investigaciones Químicas, CSIC—Universidad de Sevilla, Américo Vespucio 49,
Isla de la Cartuja, E-41092 Sevilla, Spain. E-mail: juanmab@iiq.csic.es; Fax: +34-954460565; Tel:
+34-954489560
Cyclodextrin (CD) history has been largely dominated by their unique ability to form inclusion
complexes with guests fitting in their hydrophobic cavity. Chemical funcionalization was soon
recognized as a powerful mean for improving CD applications in a wide range of fields,
including drug delivery, sensing or enzyme mimicking. However, 100 years after their
discovery, CDs are still perceived as novel nanoobjects of undeveloped potential. This critical
review provides an overview of different strategies to promote interactions between CD
conjugates and genetic material by fully exploiting the inside-outside/upper-lower face
anisotropy of the CD nanometric platform. Covalent modification, self-assembling and
supramolecular ligation can be put forward with the ultimate goal to build artificial viruses for
programmed and efficient gene therapy (222 references).
Carmen Ortiz Mellet
1
Carmen Ortiz Mellet received her PhD degree in Chemistry from the University of Seville
(Spain) in 1984, where she was appointed Tenure Professor of Organic Chemistry in 1987. In
1990, she joined the group of Professor Jacques Defaye (Centre d'Etudes de Grenoble, France)
to work in the synthesis of complex thiooligosaccharides. She came back again to Jacques
Defaye's group in 1995 pursuing synthetic and supramolecular studies on cyclodextrins. Since
1998 she is responsible for the Carbohydrate Bioorganic Chemistry Group at the University of
Seville, being promoted to Full Professor in 2008. Her research interest focuses on the study of
the interactions of carbohydrates with other molecules and biomolecules, from drugs to
enzymes, lectins and DNA. The laboratory also develops a research line on prebiotic
oligosaccharides.
José M. García Fernández
Jose Manuel García Fernández received his Doctor of Chemistry degree from the university of
Seville (Spain) in 1988. He pursued his postdoctoral research at Centre d'Etudes de Grenoble
(1990–1992 and again in 1995), where he entered the field of cyclodextrins under the
guidance of Dr Jacques Defaye. In 1996 he was appointed Tenure Scientist of the Spanish
National Research Council (CSIC) at the Institute for Chemical Research (CSIC—University of
Seville), then promoted to Senior Research Scientist (2003) and Research Professor (2006).
Since 2009 he serves as Director of this Institute He authored approximately 150 scientific
articles in peer-reviewed journals, review articles and book chapters and is co-inventor of 15
patents. His research interests cover synthetic and supramolecular aspects of carbohydrates,
with emphasis in the development of biomedical applications. Current targets include
glycomimetics as chemical chaperones for the treatment of lysosomal storage disorders,
inhibitors of the biosynthesis of glycoproteins, drug and gene delivery systems and food
products with health benefits.
2
Juan M. Benito
Juan M. Benito received his PhD degree in chemical sciences at the University of Sevilla, Spain,
in 2001 working on the development of cyclodextrin-based drug delivery systems. He pursued
his postdoctoral research at Carlsberg Laboratory, Copenhagen, Denmark (2002–2003), on
combinatorial approaches to carbohydrate receptors under the supervision of Prof. M. Meldal.
In 2004, he enrolled the Institute for Chemical Research, CSIC—University of Sevilla, where he
holds a Tenured Scientist position since 2006. Molecular recognition processes in biological
systems involving carbohydrates and their application to the development of site-specific drug
and gene delivery systems are among his scientific interests.
1. Introduction
Conventional drugs consist of a formulation of a bioactive species and a carrier , accounting
the former for most of the sophistication of the design. In the case of biomolecular drugs such
as genes , however, the role of the carrier becomes decisive in enabling the load to reach its
target to carry out its designed therapeutic function. Actually, the clinical success of gene
therapy critically depends on the use of efficient and safe delivery systems. This issue was soon
recognized and still remains a challenge nearly 25 years later. Because of their natural ability to
infect cells , viruses were the logical choice to deliver genes to the right spot. Recombinant
viruses have been constructed by replacing the genes essential for the replication phase of
their life cycles with the therapeutic genes of interest. Initial impetus in gene therapy,
provided by the use of viral delivery in the early 90s, was set back when serious problems
associated to toxicity, immunogenicity and quality scale-up production of the vectors
appeared.1 As viruses have evolved to infect cells , the immune system has evolved to fight off
what it perceives as invading pathogens. To date, the FDA has not yet approved any viralvector-based gene delivery therapeutic due to these concerns.
3
Aware of the inherent risks of viral vectors, much attention was directed to the design of
artificial (non-viral) carriers for gene delivery.2,3 This fact, together with the advent of
nanotechnology, has boosted the field within the last 15 years. Nowadays, literally hundreds of
non-viral gene vectors have been proposed,4,5 most of them falling into one of these two
categories: cationic amphiphiles 6–8 or cationic polymers (Fig. 1).9–11 Both types of
compounds self-assemble in the presence of polyanionic nucleic acids to form small particles
(lipoplexes and polyplexes, respectively) that protect from degradation and enhance cell
permeability of the gene material.12 In contrast to their viral counterparts, non-viral vectors
are, in principle, invisible to the immune system and, since they can be tailored for a particular
purpose following a ‘bottom-up’ design, there are no restrictions on the size and amount of
the cargo to be delivered.13 Also non-viral gene vectors are likely to be much easier to scale
up and produce.
There are several key problems to be solved before non-viral systems can be therapeutically
useful.14 With few exceptions, their gene delivery efficiency and selectivity are far from that of
their viral counterparts and, furthermore, their heavily charged nature does not furnish these
systems with the optimal pharmacodynamics (e.g. biocompatibility and toxicity). Improvement
in gene delivery by non-viral systems has been achieved by either covalent or supramolecular
chemical manipulation in order to facilitate at least one of the steps involved in the
transfection pathway without adversely disturbing others.15,16 For instance, combination of
the gene -condensing capability of certain vectors with the concept of receptor-mediated
endocytosis has yielded more efficient and selective gene delivery systems performing much
like ‘artificial viruses’. Likewise, ‘steric stabilization’ has been used as a means of shielding the
positively charged surface of complexes, thereby preventing their non-specific interactions
with intra- and extracellular components. Recent evidences point to significant advantages in
using formulations that include carbohydrates such as low molecular weight chitosan17 or
cyclodextrins (cyclomaltooligosaccharides, CDs) both in cultured cells and animals. The
rationale behind these designs was originally to impart stability towards biological fluids,
biocompatibility and membrane -crossing capabilities. Interestingly, the chances to interfere
and manipulate gene delivery capabilities of the vectors are much greater than initially
expected especially in the case of CDs.
Cyclodextrins are naturally occurring cyclic oligosaccharides composed of α(1→4)-linked
glucose units arising from enzymatic degradation of starch that hold a privileged position as
drug delivery and controlled drug release systems. They feature a basket-shaped topology in
which glucose hydroxyls orient to the outer space flanking the upper and lower rims, while
methinic protons (H-5 and H-3) point to the inner cavity (Fig. 2). Such structure imparts a
singular ‘inner–outer’ amphiphilic character and endows CDs with molecular inclusion
capabilities, which has been profusely exploited by the pharmaceutical industry to improve
bioavailability of poorly soluble or biodegradable drugs , to prevent undesired effects or to
enhance permeability of biological membranes . The kinetics and thermodynamics of complex
formation with non-polar guests of appropriate size and shape govern the release of the
included guest.18–20 Due to these features CDs have turned into some of the most valuable
“off-the-shelf” tools to face the challenge of the increasing number of poorly soluble drug
candidates running through clinical trials. A number of drug –CD complexes have been
marketed (basically small drugs and drug -like molecules)21–26 and applications have been
4
extended to the agrochemical ,27 cosmetic28 and food industry.29,30 But a body of evidence
also indicates that CDs might be useful for formulation improvement on biomacromolecule
delivery.31–34 This review will particularly concentrate on their applications in the field of
non-viral-mediated gene therapy, to which CDs are significantly contributing in the last decade.
2. Cyclodextrins as gene delivery efficiency enhancers
Apart from their inherent properties as nanometric containers,18 CDs ability to improve drug
bioavailability has been suggested to benefit from two additional features: (i) their membrane
absorption enhancing properties and (ii) their ability to stabilize biomolecules in physiological
media by shielding them from non-specific interactions.35,36
CDs interaction with biological membranes results in the release of certain membrane
components (e.g. cholesterol or phospholipids ) and consequently, their destabilization and
permeabilization.37 The more lipophilic CD derivatives can alter lipid distribution in vivo and
affect cell signalling through the alteration of lipid raft systems.38,39 While CD-mediated
membrane disturbing effects could be a limitation to the extended use of cyclodextrins (e.g.
native βCD is known to be parenterally nephrotoxic due to cholesterol removal from cell walls
), if appropriately tuned these systems can be exploited to change the properties of the mucus
layer , induce tight junction opening or even stimulate cellular uptake by specific mechanisms.
In addition, a number of CD derivatives have been shown to prevent aggregation in solution of
a number of proteins (e.g. human growth hormone or insulin ) and have been extensively used
to stabilize lyophilized macromolecular therapeutic formulations.32,40–43 Interestingly, CDs
have also proven beneficial for increasing the stability of oligonucleotides (ONs) against
endonucleases or even modulating undesirable side effects such as immune stimulation.44,45
The efficiency of gene delivery systems can be improved by the addition of cyclodextrin
derivatives as formulation excipients. The rational of such enhancement is however, not well
defined, probably because several of the above commented features of CDs (molecular
inclusion, membrane disturbing and macromolecule shielding) simultaneously operate. In a
seminal report, Niven and Freemann quantified a 6-fold enhancement in gene expression in rat
lung when adding βCD (1%) to the original DNA [thin space (1/6-em)]:[thin space (1/6-em)]lipid
formulations.46 The authors attributed this effect to CD–membrane permeation enhancement
capabilities in this tissue.47 This increase is modest as compared with the effect of other
additives (125-fold increase for sodium glycholate-containing formulations), but βCDcontaining formulations showed no apparent toxicity in vivo. Inspired by these results,
Roessler et al. conducted the first study on the interactions between polymer -based
transfection systems and CDs.48 Rather than noticing a direct effect on cell membrane
permeability, they observed that inclusion of CDs into the formulations resulted in particles
that were smaller, more stable and evenly distributed. In vitro assays using these particles as
functional coatings on collagen -based biodegradable membranes furnished up to a 200-fold
increase in gene expression as compared to CD-free formulations. The best performance was
achieved using sulfated anionic CDs, aiming at gradually promoting cationic polymer –DNA
complex dissociation.
The stabilizing effect that cyclodextrins impart to gene delivery systems has been observed
even for viral vectors. Croyle and coworkers observed that some neutral and cationic CD
5
derivatives enhanced adenoviral-mediated gene expression .49 They interpreted that cationic
CD derivatives interact with the negatively charged adenoviral surface preventing non-specific
interactions and facilitating their access to hard-to-transfect cells (e.g. intestinal epithelial cells
). The CD shield improves viral dispersion and bioavailability thus facilitating cellular absorption
of adenoviral vectors even after prolonged storage .50 Unfortunately, there is no direct
correlation between in vitro and in vivo performance.51
Birchall and coworkers have shown that spray-dried lipid -polycation–DNA complexes
containing dimethyl-βCD (DIMEB; Fig. 3, left) displayed significantly greater transfection
efficiency as compared to a CD-free formulation. Transmission electron microscopy (TEM )
revealed that the CD derivative substantially alters particle morphology and size distribution,
the resulting powder exhibiting excellent performance for pulmonary gene delivery.52 The
utility of cyclodextrins to enhance gene complex stability has been further demonstrated by
Jessel and coworkers53,54 by building up a multicomponent gene delivery system consisting of
transfectious DNA complexes formulated with a cationic CD, namely heptakis(6-deoxy-6pyridylamino)-β-cyclodextrin 155 (Fig. 3, right) embedded into multilayered films (e.g. poly-Llysine/hyaluronic acid (PLL/HA)).54 They showed that the efficiency of these systems to deliver
the CD-DNA complexes to the deposited cells was several orders of magnitude higher as
compared with the same architecture without CD. This tool should have a significant impact on
the development of localized gene therapies envisioned to tissue engineering.56,57
Cholesterol-containing gene delivery formulations have also benefited from the addition of
CDs. Cholesterol is determinant in regulating membrane fluidity and therefore cellular uptake.
However, due to its low solubility, just adding cholesterol to conventional lipid –DNA
formulations is not sufficient to enhance uptake. Mahendran et al. have circumvented this
problem by adding instead methyl-βCD-solubilized cholesterol, thus preventing aggregation
and stabilizing the formulation.58 In contrast to that observed for CD-free formulations, DNA is
mainly localized in the nucleus , suggesting that CD-solubilized cholesterol affects not only the
permeability of the cellular envelop, but also endosomal and nuclear membranes .
Based on the same concept, Sakurai and coworkers developed a series of cholesterolappended derivatives of schizophyllan , a fungal polysaccharide consisting of a β(1→3)-Dglucan backbone grafted with β-glucosyl residues at OH-6 once every third glucose unit,59 is
known to form very stable complexes with oligonucleotides (ONs)60,61 but with limited
membrane trespassing abilities. When using schizophyllan derivatives appended with
cholesterol moieties, antisense ON complexes displayed enhanced transfection activity, but
unfavourable complex formation kinetics at the most interesting cholesterol densities, which
impairs taking full advantage of cholesterol-mediated cellular uptake (Fig. 4). To circumvent
this drawback, the authors formulated the complexes in the presence of βCD, resulting in
improved complexation ability while preserving uptake and cell viability.62
The cell membrane -disturbing effect of CDs has been put forward in a recent report by
Aachmann and Aune describying successful gene delivery into bacteria. Plasmid and
megaplasmid formulations containing βCD derivatives resulted in the largest enhancements
(up to 4-fold as compared with CD-free formulations) after heat-shock. Since bacterial cell
walls impair the bacteria from handling large complexes or aggregates, the observed effect
6
was ascribed to the capability of the CDs to extract membrane components without lysing the
cell , thereby making it more permeable to DNA .63
3. CD-based polymers in gene delivery
Polymeric CD-containing materials have been exploited for biomedical and pharmaceutical
purposes since the 80’s.64–66 The first examples used cross-linking reagents such as
epichlorhydrin or bis(isocyanate) derivatives to obtain highly polydispersed networks in which
the CD units are connected via ether or urethane functionalities (Fig. 5A). Pre-existing
polymers 67,68 or dendripolymers69 incorporating pendant or coating CD moieties (Fig. 5B)
and polymers with integrated CDs in their backbone70 (Fig. 5C) were reported soon later.
Nowadays, the repertory of CD-based polymers includes macroscopic hydrogels and
micro(nano)particles designed for controlled or sustained drug release, molecular absorption,
tissue engineering or localized delivery of therapeutic agents.71,72
Numerous attempts to translate the benefits of CD-based polymeric structures to gene
delivery have been reported. For the purpose of this review the discussion of the results will be
divided into two separate folders depending on the location of the CD moieties in the polymer
network. The first one includes those polymeric species containing CDs in their backbone,
namely CD-embedding polymers while the second comprises pre-existing gene delivery
polymers grafted with CD derivatives, namely CD-pendant polymers .
(a) CD-embedding polymers
The application of CD-containing polymers to gene delivery73–75 was pioneered by the group
of Mark E. Davis who, encouraged by the emergence of CD-based polymers for drug delivery
and the high prospects of polycationic species as non-viral gene vectors, conceived a new class
of cationic polymers specifically designed to deliver macromolecular therapeutics. Their
synthetic strategy was based on the polycondensation of difunctionalized CD monomers
(e.g.2) in which two hydroxyls have been regioselectively replaced by cysteaminyl segments
(→3) and cationic difunctionalized co-monomers (e.g.4), in order to form a linear polymeric
chain with alternating CD and cationic units (5 in Scheme 1).70 Electrostatic-driven
complexation of the resulting cationic CD polymers (CDPs) and negatively charged pDNA
([similar]5 kpb) rendered nanometric polyplexes (polyCDplexes; 100–150 nm) featuring in vitro
cell transfection efficiency comparable to that obtained with polyethyleneimine (PEI) and
Lipofectamine™ (a commercial formulation consisting of a 75[thin space (1/6-em)]:[thin space
(1/6-em)]25 mixture of the cationic lipids DOSPA and DOPE; see Fig. 1) while preserving a
reduced toxicity.
This milestone contribution was followed by a series of reports in which Davis and coworkers
investigated the structural effects of their CDPs on gene delivery capability.76–81 The
influence of factors such as the CD size (β- or γCD, in comparison with linear saccharides)77,79
and the distribution and nature of cationic elements78,80 (their linkages, distances,76 and
relative dispositions),77 as well as the polymer size and polydispersity,79 has been examined
(Fig. 6). Structure–activity relationship (SAR) studies concluded that low molecular weight
polymers (ca. 10 kDa, degree of polymerization , DP, 5–8) with the CD units sufficiently spaced
7
from amidine cationic centers were the optimal architectures in terms of both high delivery
efficiency and low cytotoxicity (Fig. 6).81
More recently, the same authors have reported a significant improvement in delivery
efficiency by modifying the polymer endings with imidazole groups .82 Though the precise
mechanism leading to this efficiency enhancement is unclear, the authors argued that
imidazole moieties should impart buffering capacity to CDP-gene polyCDplexes that would
prove instrumental for efficient endosomal release by virtue of the “proton sponge” effect.83
Very recently Srinivasachari and Reineke have investigated a versatile approach towards linear
CD-containing cationic polymers for pDNA delivery by using Cu(I)-catalyzed azide-alkyne 1,3dipolar cycloaddition 84 of a diazido βCD derivative (6) and α,ω-dipropargylated
oligoethyleneimines (Scheme 2). The gene expression profiles of the resulting “click” polymers
(7) in HeLa (human epithelial cervical carcinoma) cells were mostly dependent on the
oligoethylenemine/CD ratio, with the polymers having longer oligoethyleneimine segments
being the better performers.85
The group of Yu and Wang reported a synthetic procedure to crosslink hydroxypropyl CDs
(HPCDs) and short PEI chains (PEI600) by reaction with carbonyl diimidazole (CDI). The
resulting polymers (8, Scheme 3) displayed high transfection efficiency in SKOV-3 (human
adenocarcinoma) cells comparable to that of bPEI (25 kDa).86 The carbamate linkages ensure
polymer bio-degradability which contributes to reduce toxicity. Slight differences in gene
delivery efficiency were observed depending on CD size (α, β or γ). Transfection efficiencies up
to 5.5 fold higher as compared with PEI (25 kDa) polyplexes were obtained with these types of
polymers in SKBR-3 human breast cancer cells in complete serum media.87 Similar βCD-PEI600
polymers were also shown to deliver genes into refractive cell lines such as cultured neurons
.88
The same authors have achieved in vitro transfection in several tumor cells by covalently
grafting βCD-PEI600 polymers to folic acid (Fig. 7), a ligand for which specific receptors are
overexpressed in several types of cancer cell lines, while maintaining a toxic profile far below
that observed for branched polyethyleneimine (bPEI; 25 kDa). Furthermore, in vivo optical
imaging showed an efficiency comparable to that of adenovirus-mediated transduction in
melanoma-bearing mice without inducing apparent toxic effects.89 Similarly, HPγCD-PEI600
polymers grafted to peptide ligands of the human epidermal growth factor receptor
(overexpressed in several breast and ovary cancer cell lines) can efficiently transfect the target
cells both in vitro and in vivo.90 Animal experiments using a therapeutic gene showed
significantly enhanced antitumor effects on tumor-bearing mice as compared to bPEI (25 kDa)
and non-targeted HPγCD-PEI600 polymers .
Amiel et al. have exploited the host –guest concept, taking advantage of the CD inclusion
capacity, to induce polyCDplex formation. They used a neutral epichlorohydrin-cross-linked
βCD polymer and cationic compounds with a structural motif having high affinity for the βCD
cavity (e.g. positively charged adamantane or cholesterol derivatives; Fig. 8).91,92 The authors
claim that the charge density of the supramolecular polymer can be finely tuned by acting on
the proportion of cationic guest thereby offering the possibility to modulate the DNA
complexing abilities. The ternary CD polymer –cationic guest–DNA formulations showed gene
8
transfection efficiencies mostly dependent on the cationic density. The best performing
formulation used a fusiogenic component and compared well with DOTAP-based lipoplexes.93
An interesting feature of CDPs is that the nanoparticles obtained upon oligonucleotide
complexation can be modified at their surface by exploiting the intrinsic CD inclusion
capabilities. Davis and coworkers have taken advantage of this property to coat preformed
polyCDplexes with functional elements that impart stability and targeting capabilities in view
of systemic applications. For instance, the inclusion of the adamantane moiety of adamantanemodified polyethyleneglycol (Ad-PEG) into CD cavities of CDP–pDNA complexes creates a steric
shield around the particles that prevents aggregation and non-specific interactions with
biological components.94 Furthermore, polyCDplexes coated with galactosylated Ad-PEG were
shown to exhibit selectivity towards hepatocytes with galactose specific membrane
receptors.95 PolyCDplexes covered with transferrin-modified Ad-PEG (Ad-PEG-Tf)96,97
transfected luciferase -encoding gene to K562 human myelogeneous leukemia cells with better
efficiency than non-targeted particles (Fig. 9).96 These Tf-targeted gene vectors have shown in
vivo transfection efficiency and selectivity towards different tumor models (murine98–100 and
primates).101 In June 2008, a targeted therapeutic based in this concept, CALAA-01 (Calando
Pharmaceuticals ) entered phase I clinical trials. The active ingredient of CALAA-01 is a small
interfering RNA (siRNA ). This siRNA inhibits tumor growth via RNA interference to reduce
expression of the M2 subunit of ribonucleotide reductase (R2). The CALAA-01 siRNA is
protected from nuclease degradation within a stabilized polyCDplex termed RONDEL™ (RNAioligonucleotide nanoparticle delivery). RONDEL is a three part delivery system: the cationic
linear CDP, the AD-PEG surface modification element to increase stability and serum half-life
and the Ad-PEG-Tf targeting ligands that assure targeting of the nanoparticles to the tissues of
interest. The study is directed to adults with solid tumours refractory to standard-of-cure
therapies and is currently recruiting participants.102 Alternative targeting ligands (e.g. cancer
cell markers and antibodies ) are being investigated.73,103,104
(b) CD-pendant polymers
An important amount of effort has been devoted to manipulate the properties of pre-existing
gene -transfecting polymers by attaching CDs. Since many early gene -delivery formulations
used commercially available cationic polymers (not purposely designed for this task), it was
reasonable that the performance of those that serendipitously worked could be engineered by
chemical manipulation to enhance their delivery efficiency, to avoid or diminish their toxicity
or to furnish them with additional capabilities (ideally all of them). In this regard,
polyethyleneimine (PEI) is a paradigmatic case, being one of the most effective artificial gene
delivery systems.105,106 But because PEI is an off-the-shelf material, it is not surprising that
its properties are sub-optimal for gene delivery. In fact, the use of PEI in gene delivery has
been hindered by its relatively high cytotoxicity.
In a hallmark report, Davis and coworkers assessed the influence that CD-grafting exerts in the
gene delivery capabilities of commercially available branched and linear PEIs.107 A series of
CD-grafted polymers were constructed by reacting controlled proportions of mono-6-O-tosylβCD with the commercial polymers . Though toxicity was alleviated, CD grafting was
detrimental for transfection efficiency as compared to naked PEI. However, co-formulation
9
with Ad-PEG conjugates significantly increased nanoparticle stability in culture media, thus
recovering much of the efficacy. In vivo experiments in mice demonstrated the potential of
PEG-shielded CD-PEI-DNA polyCDplexes for systemic gene delivery.107
The molecular inclusion capabilities of the CD units in CD-grafted PEI polymers have been
exploited also to decorate DNA polyCDplexes with targeting ligands. Pack and coworkers
synthesized a derivative of human insulin bearing a fatty acid alkyl chain that could be
accommodated into the CD cavity.108 The ternary formulation of CD-PEI, DNA and targeting
ligand furnished virtually non-toxic particles that displayed over 10-fold higher gene delivery
efficiency than PEI.
CD-grafting of PEI can be employed to promote PEI-DNA nanoparticle immobilization onto
solid surfaces. Thus, Pun and coworkers took advantage of the supramolecular interaction
between adamantane-functionalized surfaces and βCD-grafted PEI-DNA polyplexes to conceive
a system that could release transfectious particles in a controlled manner.109 Implementing a
similar concept, Ma and coworkers have described a CD-based polymeric assembly designed to
act as dual (gene and drug ) delivery system. Inclusion-driven supramolecular assembly of a
βCD-grafted PEI with poly(β-benzyl-L-aspartate) (PBLA) generated core–shell-structured
nanoparticles which a hydrophobic core that can allocate hydrophobic drugs , and a cationic
shell intended to condense DNA (Fig. 10).110 As a proof of principle, the authors determined
transfection efficiency towards osteoblast cells using both neat and drug -loaded
(dexamethasone, DMS ) particles. Though gene delivery was less efficient than that reported
for conventional PEI-based polyplexes, it is remarkable that drug loading exerted a mild
positive effect on both cell viability and gene transfer.
Grafting cationic polymers with CDs for gene delivery purposes has not been limited to PEI. For
instance, Harashima and Yui synthesized βCD-grafted poly-L-lysine (PLL) polymers by reacting
commercial PLL with 6-O-tosyl-βCD. This modification translated into an enhancement in the
cellular uptake of the corresponding polyCDplexes and improved cytoplasmic trafficking to the
perinuclear region.111 The authors reasoned that the CD moieties covering the outer part of
the polyCDplex may promote interaction with membrane components (e.g. cholesterol), thus
inducing endocytosis . Furthermore, the pronounced influence of pH in polyCDplex stability let
the authors infer that the newly generated secondary amine group upon βCD grafting may
confer “proton sponge” capabilities to the nanoassembly.
CD-grafting has also proved useful to improve the gene delivery capabilities of chitosan, a
natural polysaccharide consisting of β(1→4)-linked glucosamine units (see Fig. 1). Although
non-toxic, chitosan itself exhibits limited gene delivery capabilities and numerous alternatives
have been reported in order to improve them.17 In this context, Alonso and coworkers
recently reported that pentasodium polyphosphate-mediated cross-linking of native chitosan
with anionic CDs (e.g. sulfobutylether-βCD) furnishes nanometric particles (100–200 nm) that
were more efficient at entrapping and stabilizing pDNA than conventional chitosan
formulations. Furthermore, the resulting complexes feature enhanced cellular uptake and
expression into epithelial cells .112
From a more fundamental perspective, Liu and coworkers have recently evaluated the DNA
condensing capabilities of CD-functionalized chitosan polymers in the absence and in the
10
presence of hydrophobic components that interact with the cationic polymer (e.g.
adamantane-functionalized pyrene, Fig. 11).113 The authors noticed that the DNA-condensing
capabilities of CD-functionalized chitosan polymers were considerably enhanced in the
presence of the hydrophobic components, attributing such enhancement to cooperativity
between electrostatic and hydrophobic interactions.
4. CD-based polyrotaxanes in gene delivery
Rotaxanes , a type of mechanically interlocked system consisting of a cyclic molecule threaded
by an axial species, have been revealed as an unflagging source of molecular machines and
stimulus responsive nanosystems exclusively limited by chemists’ creativity. The cyclic
component of a (poly)(pseudo)rotaxane can slide and/or rotate along the axis, eventually
endowing the supramolecular construct with features that are not possible in conventional
covalent architectures. As a paradigmatic wheel-like species, CDs have played a major role in
this field.114Rotaxanation of a polymeric species with CDs might translate into major changes
regarding for instance hydrophilicity, environmental shielding, flexibility and/or functional
coating. Furthermore, previous studies have clarified that the mobility of ligands linked to the
cyclic compounds is closely related to enhancing multivalent interaction with biological
systems.115 This concept is nowadays mastered for a variety of biomedical applications116
and yielded, in the last few years, some of the most sophisticated systems envisioned for DNA
complexation and gene delivery purposes.22,117,118
Kissel and coworkers described the first example in which rotaxanation of a linear polycationic
polymer with CDs contributed to improve gene delivery efficiency.119 These authors had
previously reported interesting transfection capabilities for branched polyethyleneimine-polyε-caprolactone–polyethyleneglycol (9, bPEI-PCL-PEG) block copolymers (Fig. 12).120 However
they also noted that, as a consequence of H-bonding-driven collapse between PCL and bPEI
blocks, copolymer solubility and bPEI-pDNA binding were inherently limited. To circumvent
this problem they proposed a creative solution consisting on threading αCD units through the
polymer chain (10, Fig. 12). The higher affinity of PCL segments for the αCD cavity as compared
to PEG and PEI blocks results in their preferential shielding, thus benefiting polymer solubility
and bPEI interaction with pDNA.
Gel retardation and visualization experiments with the intercalating agent ethydium bromide
showed that the pDNA-binding capability of the copolymer upon rotaxanation improved to be
as efficient as that observed for bPEI (25 kDa). Furthermore, transfection efficiency of the
corresponding CD-based rotaxane-pDNA complexes (rotaCDplexes) in mouse embryonic
fibroblasts 3T3 cells was in the same order of magnitude as that of bPEI-based polyplexes but
with a 100-fold lower toxicity. The virtually neutral surface potential (ζ potential) of these
rotaCDplexes did not impair efficient cell uptake and is probably responsible for the absence of
toxic effects.
Li and coworkers developed a novel strategy to build up series of supramolecularly assembled
cationic
polyrotaxanes
using
βCD
and
polyethyleneglycol-polypropyleneglycolpolyethyleneglycol (11, PEG-PPG-PEG) triblock copolymer (pluronic copolymer ). The synthetic
scheme consisted on a three-step sequence involving (i) threading of βCD units along the
amine -terminated pluronic polymer , (ii) capping the polymer ends with bulky 2,4,611
trinitrobenzenesulfonate (TNBS) stoppers (→13), and (iii) grafting oligoethyleneimine (OEI)
branches onto the βCD moieties using labile carbamate linkages (→14) (Scheme 4).121 The
authors hypothesized that the higher stability of PPG–βCD as compared to PEG–βCD
complexes122 limit the number of βCD units threaded on the PEG-PPG-PEG polymer (average
13 βCD units per 2.9 kDa polymer in this case), providing free space for the βCD units to move
along the polymer and thereby facilitating optimal OEI–pDNA interactions.
Fluorescent titration experiments indicated that the PEG-PPG-PEG/βCD-OEI polyrotaxanes 14
could completely condense pDNA at N/P values (ratio between protonable amino groups in
the vector and negative phosphate groups in the plasmid ) ≥ 2. The length of the OEI chain size
(p = 0, 4, and 8) and their density played little influence on pDNA condensing efficiency of the
polyrotaxanes.123 At N/P 10, 150–200 nm nanoparticles were obtained, slightly larger than
those measured for PEI. Gene delivery in human embryonic kidney HEK-293 cells also featured
a very similar profile, though in this case polyrotaxanes grafted with the longer OEI branches (p
= 8 in Scheme 4) behaved systematically more efficiently than the others, rivalling PEI
polyplexes even in the presence of serum.121
This promising result prompted the authors to obtain a detailed map on how the polyrotaxane
structure controls gene expression . They have reported that polyrotaxanes constructed by the
same strategy from αCDs and random PEG-PPG polymers yield consistently smaller
rotaCDplexes (av. 100–200 nm). Polyrotaxanes grafted with short OEI chains exhibited far less
cytotoxicity than bPEI (25 kDa), though their transfection efficiency was not always optimal.
Polyrotaxane efficiency to mediate pDNA expression was assessed in a variety of cell lines
including HEK-293, African green monkey kidney cells COS-7, Syrian hamster kidney fibroblasts
BHK-21, SKOV-3 and human uterine sarcoma MES-SA cells . Delivery efficiency in the absence
of serum was similar to that obtained with bPEI (25 kDa), eventually surpassing it for MES-SA
cells .124 However, in the presence of serum performance showed a strong cell type
dependence.
More recently, the same authors have completed their SAR mapping by exploring the gene
delivery capabilities of two additional series of polyrotaxanes consisting on OEI-grafted αCD
units threaded on PEG polymers 125 and PPG-PEG-PPG block copolymers ,126 respectively.
Though none of these series significantly improved overall transfection efficiency, some
important conclusions were inferred. On one hand, αCD-PEG polyrotaxanes were shown to
form slightly smaller particles upon pDNA complexation and their transfection efficiency was
not significantly affected by the presence of serum.125 This might indicate that the higher CD
mobility along the PEG polymer contributes to a tighter pDNA complexation and more efficient
environmental isolation. On the other hand, polyrotaxanes constructed by threading αCD units
on a PPG-PEG-PPG (15), were shown to induce sustained gene expression in HEK-293 cells (Fig.
13),126 in contrast with that observed for PEI polyplexes and for the inversely supported
polyrotaxanes based on PEG-PPG-PEG.121 The authors did not provide an explanation for this
differential performance, though these results call the attention on how subtle structural
modifications can influence gene delivery efficiency.
The CD–polyrotaxane architecture is particularly well suited for the design of gene vectors
capable of liberating the oligonucleotide load at the intracellular space after a cell -dependent
12
chemical input.116 Aware of the utmost importance that timely transfecting complex
dissociation has on overall gene delivery efficiency, Yui and coworkers ingeniously exploited
both CD mobility and polyrotaxane dissociation to envision a CD-based nanosystem featuring
controlled pDNA release capabilities.127 They synthesized biocleavable polyrotaxane 18 by
threading cationic αCD derivatives (dimethylaminoethylcarbamoil-grafted αCD, DMAEC-αCD)
onto PEG chains (4 kDa) that were capped with benzyloxycarbonyl tyrosine via disulfide
linkages (Scheme 5). The resulting polyrotaxanes efficiently condense pDNA into <200 nm
nanoparticles .128 The rational of their design relied on the fact that the higher intracellular
reducing potential should promote disulfide cleavage, therefore favouring CD dethreading and
pDNA release.129 As proof of concept, they demonstrated that 10 mM dithiotreitol (DTT)
slowly degraded polyrotaxane–pDNA nanoparticles . Moritorization by confocal laser scanning
microscopy (CLSM ) of 18–pDNA nanoparticle trafficking in the mouse embryonic fibroblast cell
line NIH3T3 revealed rapid endosomal escape (faster than that of PEI-based polyplexes) and
selective localization of the pDNA cargo inside the nucleus after 90 min.127,130,131
Variation of the number of CD units threaded per polymer chain as well as the extent of
DMAEC grafting allowed the authors to optimize pDNA packing and realising capabilities.
Ethidium bromide intercalation assays showed that pDNA compaction was more efficient for
polyrotaxanes displaying large numbers of DMAEC groups (ca. 5 per CD). However, the
transfection levels were higher for systems having lower density of cationic chains (ca. 2–3
DMAEC groups per CD). The authors attributed this observation to the much faster pDNA
release from the corresponding rotaCDplexes, thus highlighting the importance of
programming nanoparticle dissociation for efficient DNA delivery.132
Rotaxanation of cationic polymers using CDs in order to shield or reduce their charge density
has been also investigated.133 Although intuitively more straightforward to tackle cationic
polymer toxicity drawbacks, this strategy is handicapped due to the less efficient threading of
CDs through cationic axis. Linear PEI and PLL threading with CDs can only be efficiently
achieved at elevated pH that prevent from protonation of the amine groups .134,135 Bearing
in mind the inherent toxicity of conventional cationic polymers , Yui and coworkers have
investigated linear PEI (lPEI)-γCD polypseudorotaxanes as safer gene carriers.136 Though these
supramolecular constructs condensed pDNA less efficiently than naked lPEI (22 kDa), their far
lower toxicity and the better cellular uptake of the corresponding rotaplexes fully
compensated, achieving similar gene expression levels than PEI-based polyplexes in NIH3T3
cells .
Inspired by Schneider's design of switchable DNA intercalating agents based on anthranylfunctionalized CDs,137 Liu and coworkers have developed an original and conceptually distinct
approach towards DNA -binding polypseudorotaxanes by threading antharyl-modified cationic
βCD precursors (Fig. 14) on amine -terminated PPG chains (19, av. 10 βCD units per 2 kDa PPG
chain). Initially envisioned as DNA-reactive supramolecular assemblies 138,139 rather than
gene vectors, fluorescence titration and atomic force microscopy (AFM ) demonstrated the
capability of polypseudorotaxane 19 to condense DNA into small nanoparticles (ca. 100 nm).
Compactness is probably achieved by cooperative contributions of cationic and aromatic
elements to the binding process.140
13
The anthranyl-grafted polypseudorotaxanes proved useful to promote double-stranded DNA
wrapping around single wall carbon nanotubes (SWCNs) and mediate photoinduced DNA
cleavage.141 A more sophisticated design was further developed for specific application in
gene delivery. It consists of a 2-dimensional polypseudorotaxane (23) constructed by threading
ω-aminohexylamino βCD on a PPG polymer backbone, followed by complexing cucurbit[6]uril
units (20) on the branches of the modified CDs (Fig. 15).142,143
Interestingly, by adjusting the cucurbit[6]uril ratio, DNA condensing capabilities can be finely
tuned, reaching the highest efficiency at 70% coverage of the aminohexyl arms as
demonstrated by ethidium bromide intercalation assays. Further investigations of the
electrophoretic properties of simpler models of this type of polypseudorotaxanes allowed
disclosing the polymer length for optimal DNA condensing efficiency.144 Unfortunately, data
on cell transfection efficiency for the corresponding rotaplexes are not available to date.
5. CD-based dendrimers and dendripolymers in gene delivery
The intrinsic polydispersity and random conformation of cationic polymers and polyrotaxanes
represents an obstacle for structure–activity relationship (SAR) studies and has raised some
concerns about the reproducibility of their preparations, threatening their regulation by the
legal authorities. In the search for better defined gene delivery systems, dendrimers represent
an interesting alternative.145,146 Because of their predictable and tailored structure,
dendrimers have been long exploited in biomedical fields.147,148 The chemical resemblance
between cationic dendrimers and some of the most successful gene transfecting cationic
polymers has further stimulated their prospect as alternative non-viral gene vectors. Not
surprisingly, DNA can wrap around these dendrimers , promoting collapse of DNA into more
compact supramolecular structures called dendriplexes, where the plasmid is protected from
the environment.149 Both CD-coated pre-existing dendrimers (Fig. 16A) and de novo
constructed CD-centered dendri(poly)mers (star-shaped dendri(poly)mers, Fig. 16B) have
shown to enhance the transfecting capabilities of the parent structures. Relevant examples of
each type of architecture are commented hereinafter.
(a) CD-coated dendrimers
Among cationic dendrimers with gene delivery capabilities, the polyaminomethylene core
(PAMAM, Fig. 17) holds a prominent position, which is adscribed to the ordered cationic
envelop of PAMAM dendrimers and the pH buffering properties of their tertiary amino groups
. As first demonstrated by Szoka and coworkers, nanoparticulated dendriplexes obtained by
mixing DNA and commercial PAMAM dendrimers exhibit remarkable transfection efficiency
towards a range of cell lines.150 Controlled degradation of PAMAM dendrimers has been
shown to enhance transfection efficiency; this is the working principle of SuperFect™, a
commercially available gene vector (Quiagen).151 Alternatively, peripheral functionalization of
PAMAM with cyclodextrins has proved to be a versatile strategy to optimize pDNA
complexation and to modulate the transfecting capabilities of the resulting
dendriCDplexes.152
Previous reports stated that the efficiency of homogeneous PAMAM in gene delivery is
circumscribed to relatively high dendritic generations (>G3) for which toxicity is a
14
concern.153,154 To circumvent this drawback, Uekama and coworkers exploited the biocompatibilizing and membrane merging capabilities of CDs.155 Covalent grafting of CDs onto a
low generation starburst PAMAM (G2, 16 primary amino groups ) by nucleophilic attack of
PAMAM primary amino groups to O-6-monotosylated CDs (24) (Scheme 6) furnished CDPAMAM conjugates with similar pDNA binding properties than the naked dendrimer , but
boosted transfection potency. In the case of αCD, up to a 100-fold efficiency enhancement as
compared to G2 PAMAM alone or physical mixtures containing αCD was reported. The authors
explained the CD type-dependent performance of the conjugates in terms of the capability of
the differently-sized CDs to interact with membrane components. To fulfil a complete
structure–activity map, they investigated the optimal PAMAM dendrimer generation (G2 to
G4, up to 64 primary amino groups )156 and the CD-coating density (av. 1 to 5 αCD units per
dendrimer ).157 In one hand, they observed that higher dendrimer generations yielded more
compact dendriCDplexes, but simultaneously increased toxicity. On the other hand, the more
heavily αCD-grafted dendrimers (ca. 5 CD units per dendrimer ) featured significantly higher
membrane disturbing capabilities, but also translated into undesired toxic effects. The
combined conclusions led the authors to propose a privileged candidate consisting in a G3
PAMAM dendrimer coated with av. 2.4 αCD moieties per dendrimer unit. This conjugate
demonstrated a high efficiency to deliver short hairpin RNA expressing pDNA (shpDNA)158 and
siRNA into cells .159,160
The above G3 PAMAM-αCD carrier promoted in vivo gene expression in mice more efficiently
than the corresponding PAMAM dendrimer in several organs (spleen, liver, kidney, and
lung).157 In an attempt to impart tissue-specificity, ternary conjugates incorporating
biorecognizable carbohydrate ligands were prepared. Thus, α-mannosides161–163 and αgalactosides164 were attached via thiourea linkages to αCD-PAMAM vectors to implement the
glycoside mediated transfection concept (“glycofection”) first proposed by Monsigny and
coworkers (Scheme 7).165 Although heavy glycosilation of αCD-PAMAM conjugates severely
decreased DNA binding capabilities, moderately glycosylated conjugates (av. 3–5 and 10 sugar
units per dendrimer for G2 and G3, respectively) systematically showed enhanced gene
delivery capacity even in the presence of serum.166 However gene expression was not
significantly dependent on whether or not sugar -specific receptors were expressed at the cell
surface , thus pointing to intracellular events rather than to cell -specificity as the basis of the
experimental observations. This is consistent with several reports by Monsigny and coworkers
noting that glycosylation of PEI affected intracellular trafficking and nuclear localization rather
than uptake.167–169
These counterintuitive results, together with the low affinity that glycosylated CD-PAMAM
conjugates exhibited for the specific lectins,161 led Arima and Motoyama hypothesize about
the suitability of the aromatic tether originally installed between the dendritic core and the
saccharidic antennae to allow efficient recognition by specific receptors at the cell membrane .
Actually, lactosylated αCD–PAMAM conjugates exhibited a marked difference in gene transfer
activity when comparing cells expressing or not the corresponding lactose/galactose-receptor
(asialoglycoprotein) in their membrane .152 Gene expression was suppressed when the
experiments were carried out in the presence of an excess of soluble competing ligands,
thereby supporting the existence of a receptor -mediated internalization process.
15
Besides PAMAM, other CD-coated dendrimers have been recently investigated. For instance,
Tang and coworkers have described a low generation polypropyleneimine (PPI) dendrimer
grafted with βCD units through biodegradable carbamate linkages (29 in Scheme 8).170 Low
generation PPI dendrimers were already known to induce high levels of gene expression in a
variety of cells .171 Interestingly the newly reported βCD-PPI conjugate 29 surpassed naked
PPI by a factor of 100 when assayed in COS-7 cells .
The ensemble of data on CD–dendrimer conjugates for gene delivery evidences the existence
of two effects that must be counterbalanced: increasing the number of CD coating units
benefits cellular uptake but neutralization of amino groups during conjugation is detrimental
for DNA binding . In an attempt to overcome this dilemma, Marsura and coworkers outlined a
strategy consisting on the use of protonable guanidine tethers. They synthesized a
carbodiimide-linked CD dimer via aza-Wittig-type coupling reaction of 6-monoazide (30) and 6monoisothiocyanate-βCD (31) building blocks. Addition of ammonia to the heterocumulene
group gave the guanidine dimer that was finally reacted with ethylenedibromide to furnish the
cationic βCD-tetrapod 33 (Scheme 9).172 Though this example is limited to a G0 core, the
concept might be general for the assembly of CD pre-coated dendrimers . Association of
guanidinylated tetrapod with siRNA and DNA and cellular uptake of the complexes were
demonstrated by capillary electrophoresis and fluorescence microscopy , respectively.173
(b) CD-centred dendri(poly)mers and star-shaped dendrimers
Exploitation of CDs as central dendritic cores for installation of functional elements has
provided excellent tools for the investigation of multivalent interactions involved in biological
recognition processes.174–178 Not surprisingly, this strategy has also showed significant
promise in the elaboration of artificial gene vectors. Thus, Li and coworkers synthesised a
series of low molecular weight oligoethyleneimine (OEI)-grafted αCDs (34) using biodegradable
carbamate linkages (Scheme 10). The influence of OEI size and branching and the extent of CD
grafting on DNA complex formation, nanoparticle size, cytotoxicity and gene delivery was
assessed.179 The best performing OEI-αCD conjugates (statistically grafted with branched 14mer OEI) rivalled with commercial PEI (25 kDa) in terms of transfection efficiency, but featuring
a more favourable cytotoxic profile.
The concept was further extended to the preparation of a series of βCD-centred star-shaped
cationic dendripolymers by sequential radical polymerization with dimethylaminoethyl
metacrylate (DMAEMA) and oligoethyleneglycol metacrylate (Scheme 11).180 These
dendripolymers (36) formed 100–200 nm nanoparticles in the presence of pDNA that exhibited
higher transfection efficiency and lower toxicity than conventional poly-DMAEMA
homopolymers . Little, if any, differences in the performance of DMAEMA-grafted βCD and the
above described OEI-grafted αCD dendrimers were noticed.
The rather unselective chemical activation of the CD core in the above commented examples
results in heterogeneous products. Xiao and coworkers have implemented a more efficient
strategy that ensures functionalization at all (21) positions of βCD.181 Previous synthetic
strategies towards this goal were unsuccessful due to the low solubility of reagents and by16
products in the required solvent for CD core activation . The use of N-methylpyrrolidine (NMP)
as solvent is compatible with the huge excess of 2-bromoisobutyryl bromide (4 eq.) required to
ensure complete functionalization. In these conditions they obtained the homogeneously
peracylated analog of compound 35 (Scheme 10) in a remarkable 90% yield.181 Linear
polymerization of this precursor with a set of different cationic methacrylate building blocks
furnished the corresponding 21-armed βCD-centred dendripolymers (37 in Fig. 18). The most
efficient
dendripolymer
in
terms
of
gene
delivery
capabilities,
bearing
poly(dimethylaminopropylamine) head groups , favourably compared with PEI, but was much
less cytotoxic.182
Apart from “classical” CDs (α, β or γ), also large ring cycloamyloses (CAs) have been employed
as scaffolds for assembling cationic elements. Keeping in mind that the CA characteristic helical
conformations might resemble that of supercoiled DNA strains,183 Akiyoshi and coworkers
investigated their potential for gene delivery.184 For such purpose they covalently grafted
spermine, a natural tetraamine with excellent oligonucleotide binding capabilities, onto CA (19
kDa) via carbamate linkers using a similar strategy to that reported by Li and coworkers.179
Spermine-grafted CAs condensed pDNA into ca. 250 nm particles that proved to be as efficient
as PEI-pDNA polyplexes promoting transfection. Furthermore, spermine-grafted CA
systematically showed greater transfection efficiency than the corresponding linear amylose
conjugates, supporting the favourable contribution of CA helical structure for pDNA binding
and delivery.
6. Monodisperse CD-scaffold vectors in gene delivery
While the star-shaped dendri(poly)mers might represent a step forward in the bottom-up
design of CD-based artificial viruses, they still possess an essentially disperse nature, which
handicap both fundamental studies and applications. Very recently, several groups have
turned their attention to the development of monodisperse CD derivatives that could selforganize in the presence of DNA and deliver it into cells .185 This approach critically depends
on the development of efficient methods to manipulate the topology and recognition features
of CDs with the environment, which represents a considerable challenge for synthetic organic
chemists. The higher accessibility of the primary hydroxyl groups (OH-6) facilitates
homogeneous functionalization at the narrower rim of CDs, which has been used to create
different types of homogeneous polycationic CD conjugates. Thus, O'Driscoll and Darcy
reported that βCD derivatives bearing alkyl and arylamine antennae on their primary rim can
complex genes and moderately mediate transfection in COS-7 cells .186
Yannakopoulou and coworkers have demonstrated that per-(C-6)-guanidino-CDs (39),
prepared by regioselective bromination of the commercial α, β and γ CDs at the primary
positions (→38) followed by nucleophilic displacement with azide anion, reduction , and
guanidinylation of the resulting amino groups (Scheme 12), tightly bind phosphorylated
substrates with a much greater efficiency than per-(C-6)-amino-CDs. Most interestingly, the
guanidino-CDs induced condensation of calf thymus DNA into nanoparticles (CDplexes) in
which the double helix was inaccessible to the intercalating agent ethydium bromide.187 The
same authors have elaborated a set of guanidinioalkylamino-CDs (40, Scheme 12) that, in
addition to improved DNA binding avidity, exhibited cell -penetrating capabilities which was
17
ascribed to their resemblance to membrane -permeable polyarginine-type peptides .188–190
The transfection efficiency of these vectors in human embryonic kidney HEK 293T cells
surpassed that of the commercial cationic lipid formulation Lipofectamine 2000™.191
Reineke and coworkers took advantage of the copper(I)-catalyzed azide–alkyne cycloaddition
reaction84 between the per-(O-2,O-3)-acetylated heptaazide 26 and acryloyl amide derivatives
to synthesize a family of polycationic βCD click clusters (27) bearing seven linear OEI branches
with variable, but controlled, number of protonatable amino groups (Scheme 13).192 Agarose
gel electrophoresis , DLS and TEM experiments revealed that the click CD-polycations
complexed pDNA by forming nanoparticles (CDplexes; av. diameter 80–130 nm) in which the
genetic material was protected from nuclease degradation . Transfection experiments towards
immortal human cervical cancer HeLa cells and rat heart H9c2 cells evidenced that the gene
expression efficiency increased with the length of the OEI chains. Optimal transfection
efficiency was reached for derivatives incorporating 4 or 5 protonatable amino groups per
chain (n = 3 or 4, respectively, in Scheme 12), with expression levels that paralleled that of the
commercial cationic polymer Jet-PEI™ or the cationic dendrimer Superfect™ but featuring a
much less toxic profile in both cell lines.
The above examples illustrate the suitability of CDs as molecular platforms for the installation
of cationic groups with a precise spatial orientation and the potential of the resulting fully
homogeneous compounds as gene vectors. Most interestingly, the rim anisotropy of the
basket-shaped CD structure further allows accessing compounds with segregated cationic and
lipophilic domains for which, according to the “facial amphiphilicity” concept, biomimetic selfassembly and gene delivery properties could be expected.185,193–197
In a pioneering work, Darcy and coworkers took advantage of the differential chemical
reactivity between the primary and secondary hydroxyls of CDs to report the first examples of
polycationic amphiphilic CDs (paCDs).198 They implemented a successful route to synthesize
amphiphilic CDs that started from the known per-(C-6)-bromoCDs (38).199 Nucleophilic
displacement of the bromo groups by fatty mercaptans and reaction of the secondary
hydroxyls with ethylene carbonate in basic medium afforded the key intermediate 45, for
which self-assembly properties in water had been previously demonstrated (Scheme 14).200
The generated terminal hydroxyls were then converted into amino groups by a reaction
sequence involving iodination , azide substitution and reduction (→46). The polycationic
amphiphilic CDs thus resulting were shown to entrap pDNA by forming CDplexes that
transfected COS-7 and human hepatocellular liver carcinoma Hep G2 cells with comparable
efficiency and toxicity profiles to Lipofectamine 2000™-based lipoplexes.201,202
The above synthetic scheme does not warrant the homogeneity of the final compounds.
Nevertheless, the authors observed a correlation of the gene delivery capability of the vectors
with the length of the hydrocarbon chain. The work has the merit to foresee the unique
opportunities that paCDs offer for chemical tailoring and structure–activity relationship studies
in gene delivery, an indispensable requisite towards the development of artificial viruses.
18
Esterification of CDs with long-chain acyl anhydrides in the presence of DMAP has been found
to ensure homogeneous products,203,204 thus opening a very convenient route to
monodisperse multi-head, multi-tail facial amphiphiles . Both possible orientations of the
putative cationic and lipophilic groups onto the CD macrocycle are conceivable, namely the
skirt-type (Fig. 19A) or the medusa-type arrangement (Fig. 19B).205 Díaz-Moscoso et al. have
shown that the polyamino amphiphilic CD (paCD) derivative 48, accessible in only four steps
from commercial βCD (Scheme 15), already formed stable CDplexes with pDNA that fairly
promoted gene expression in the murine hepatocyte BNL-CL2 cell line and human nasopharynx
carcinoma KB cells , but at much lower rate than polyplexes prepared from branched PEI (bPEI,
25 kDa).203 Most importantly, compound 48 can serve as a pivotal intermediate for further
optimization through chemical elaboration.206 Inspired in the mechanisms of phosphate
anion reversible recognition in Nature, which imply cooperative electrostatic and hydrogen
bonding interactions,207,208 and keeping in mind the proven hydrogen bond donating
capabilities of pseudoamide groups ,209,210 a belt of thiourea segments was inserted in the
structure by multiple amine -isothiocyanate coupling reaction (e.g., →49).211–214 This
structural modification boosted gene delivery efficiency by two orders of magnitude,
paralleling that of bPEI and illustrating the strong potential of diversity-oriented synthesis of
homogeneous gene vectors to correlate modifications at the atomic level with delivery
efficiency.
The same authors have further evaluated the influence of factors such as the density and
arrangement of cationic groups and thiourea H-bond donor centres, the flexibility of the
linkers or the length of the lipophilic chains in pDNA complexation and pDNA complex stability,
cytotoxicity and gene expression .215 Transfection efficiencies that surpassed by ten-fold
those of bPEI and JetPEI™ have been achieved for BNL-CL2 and COS-7 cell lines using paCDs
presenting a dendritic arrangement of cationic elements, e.g.50, while preserving much lower
cytotoxic profiles (Fig. 20).
Transmission electron microscopy (TEM ) evidenced the small size (av. diameter 40 nm) and
homogeneous distribution of CDplex formulations from 50 (Fig. 21). The snail-like
ultrastructure observed at higher magnification was attributed to alternating lamellar
arrangements of paCDs and the pDNA molecule. Rather small particle size and efficient
transfection were observed even in the presence of 10% serum.
Aiming at exhaustively mapping structure–activity relationships in paCD-mediated gene
delivery, the same authors have investigated several other paCD prototypes. For instance
Ortega-Caballero et al. synthesized a series of paCDs with an inverted orientation of the
cationic and non-polar domains on the βCD macrocycle .203 Sequential per-2,3-O-allylation of
the transiently O-6-protected βCD 51, followed by hydroboration (→52), mesylation of the
generated primary hydroxyls (→53) and substitution by cysteamine furnished a fully symmetric
medusa-shaped multifunctional platform 54 (Scheme 16). By appropriately choosing the length
of the acyl chains installed at the primary rim and finely adjusting the phosphate binding
avidity of the functional elements at the secondary rim, gene vectors that behaved as
efficiently as the skirt-type counterparts in transfection experiments using BNL-CL2 cells were
obtained. A complementary strategy, that exploits photochemical addition of
19
mercaptopropionic esters to per-2-O-allylated βCD derivatives, has been reported by Darcy
and coworkers (Scheme 17).216
Alternatively, the copper(I)-catalyzed azide–alkyne coupling reaction84 has been explored to
build CD-based facial amphiphiles . Méndez-Ardoy et al. reported a series of polycationic
amphiphilic click clusters in which the triazol segments were either directly linked to the
primary C-6 carbon of the βCD core (rigid clusters ) or separated by an acetamidocysteaminyl
spacer (flexible clusters ; Fig. 22).217 In both series, dendritic tetradecaamino derivatives were
much more efficient at compacting pDNA and protecting it from degradation by nucleases
than heptavalent analogues or non-amphiphilic conjugates. Unexpectedly, the more rigid
cluster proved to be the most efficient at promoting gene transfection in Chinese hamster
ovary CHO-k1 cells , featuring transfection efficiency comparable to Lipofectamine 2000™.
The mechanisms at work for cell internalization of CDlexes obtained from paCDs have been
recently investigated using the rhodamine-labelled derivative 60 (Fig. 23).218 CDplex uptake
and transfection efficiency in African green monkey kidney epithelial Vero cells was monitored
in the presence of selective inhibitors of the most common internalization routes, showing
that the largest fraction of complexes was taken up via clathrin-dependent endocytosis (CDE).
Interestingly, this fraction is less relevant for transfection. The smaller fraction internalized via
clathrin-independent endocytosis (CIE) is predominantly responsible for gene expression ,
which is similar to that reported for PEI-based polyplexes.219 However, the authors explicitly
avoided generalization of the conclusions to other paCDs or cell lines.
7. Conclusions and perspectives
In this review, we covered recent exciting reports regarding the use of cyclodextrins in nonviral
gene vector design. So far, most of the examples exploit the transfection enhancing
capabilities of CDs to improve the characteristics of pre-existing cationic polymers or
dendrimers , generally ascribed to their capacity to permeabilize cell membranes by affecting
cholesterol distribution. But there are many other systems that are being created. The intrinsic
inclusion capabilities of CDs let control the conformational properties of polymeric chains
through rotaxanation, thereby improving interactions with polynucleotides . The nanometric
cavity remains accessible in polyCDplexes, opening the door to surface coating through
supramolecular interactions. Most interestingly, the anisotropy of the molecule allows
preorganizing a variety of functional elements (for self-assembling, DNA binding , targeting or
visualization, for instance) with a precise spatial orientation.220
The effectiveness of the best cyclodextrin -based gene delivery systems currently developed,
as for other nonviral gene vectors, remains orders of magnitude poorer compared with viral
vectors. Nevertheless, the possibility to combine covalent and supramolecular approaches
offers new venues for the design of tailor-made artificial viruses. Keeping full structural control
by the implementation of imaginative synthetic strategies will be critical for those channels. In
addition, chemical control of architecture might be exploited for the development of CD-based
nanovehicles for therapeutic siRNA , thereby enhancing their potential for medical
applications.221 The CD polymer -containing system CALAA-01 is the first representative of
this family that enters clinical trials ten years after the concept was first launched.104 With a
20
growing understanding of the CD-based gene delivery mechanisms, it is likely that several
others will follow.
In addition to their “nanometric platform” and “supramolecular host ” character, cyclodextrins
, and particularly β-cyclodextrin units, provide a unique tool to modulate cellular cholesterol in
living cells .37 This ability might be optimized in vector design to facilitate the passage through
biological membranes . The CD commercial availability, easy and relatively inexpensive
synthesis of appropriate derivatives, facile purification , robustness and stability,
biocompatibility, lack of immunogenicity and safety match important criteria for non-viral
vectors.222 Yet, improvements on the theoretical understanding of the DNA packaging
process and physico-chemical characterization of the corresponding CDplexes, as well as much
more data on the in vivo behaviour of the different CD-based gene vectors reported, are
necessary. In all, there should be plenty of room for high level research connecting
cyclodextrin and gene therapy through interdisciplinary approaches involving chemists,
physicists and biologists.
21
Notes and references
1. S. Nayak and R. W. Herzog, Gene Ther., 2010, 17, 295
2. D. J. Glover, H. J. Lipps and D. A. Jans, Nat. Rev. Genet., 2005, 6, 299
3. T. Niidome and L. Huang, Gene Ther., 2002, 9, 1647
4. For a recent comprehensive survey on non-viral gene delivery, see: M. A. Mintzer and E. E.
Simanek, Chem. Rev., 2009, 109, 259
5. For a recent review on ON delivery technologies, see: G. De Rosa and M. I. La Rotonda,
Molecules, 2009, 14, 2801
6. R. Srinivas, S. Samanta and A. Chaudhuri, Chem. Soc. Rev., 2009, 38, 3326 RSC
7. I. S. Zuhorn, J. B. F. N. Engberts and D. Hoekstra, Eur. Biophys. J., 2007, 36, 349
8. W. L. Li and F. C. Szoka, Pharm. Res., 2007, 24, 438
9. T. G. Park, J. H. Jeong and S. W. Kim, Adv. Drug Delivery Rev., 2006, 58, 467
10. D. Putnam, Nat. Mater., 2006, 5, 439
11. M. Thomas and A. M. Klibanov, Appl. Microbiol. Biotechnol., 2003, 62, 27
12. M. A. Behlke, Mol. Ther., 2006, 13, 644
13. E. Mastrobattista, M. A. E. M. van der Aa, W. E. Hennink and D. J. A. Crommelin, Drug
Discovery Today: Technol., 2005, 2, 103
14. E. Wagner, Pharm. Res., 2004, 21, 8
15. H. Akita and H. Harashima, Expert Opin. Drug Delivery, 2008, 5, 847
16. B. Demeneix, Z. Hassani and J.-P. Behr, Curr. Gene Ther., 2004, 4, 445
17. S. Mao, W. Sun and T. Kissel, Adv. Drug Delivery Rev., 2010, 62, 12
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Deliv. Sci. Technol., 2008, 18, 303
203. F. Ortega-Caballero, C. Ortiz Mellet, L. Le Gourriérec, N. Guilloteau, C. Di Giorgio, P.
Vierling, J. Defaye and J. M. García Fernández, Org. Lett., 2008, 10, 5143
204. A. Díaz-Moscoso, P. Balbuena, M. Gómez-García, C. Ortiz Mellet, J. M. Benito, L. Le
Gourriérec, C. Di Giorgio, P. Vierling, A. Mazzaglia, N. Micali, J. Defaye and J. M. García
Fernández, Chem. Commun., 2008, 2001 RSC .
205. While the terms “skirt-shaped” and “medusa-shaped” have been used in the literature for
amphiphilic CDs bearing lipophilic tails at either the secondary or the primary hydroxyls,
respectively, they are not very intuitive when the hydrophilic segments consist themselves in
relatively long antennae. Actually, CD derivatives bearing long chains at both the primary and
secondary rims have been termed, generically, bouquet-shaped CDs. See: E. Bilensoy and A.
Hincal, Expert Opin. Drug Delivery, 2009, 6, 1
206. The amino groups in cysteaminyl CD derivatives have been previously shown to be
particularly apt to participate in nucleophilic addition reactions , even in hyperbranched
environments. See ref. 177 and 178.
207. A. K. H. Hirsch, F. R. Fischer and F. Diederich, Angew. Chem., Int. Ed., 2007, 46, 338
208. E. A. Katayev, Y. A. Ustynyuk and J. L. Sessler, Coord. Chem. Rev., 2006, 250, 3004
209. J. L. Jiménez Blanco, P. Bootello, C. Ortiz Mellet, R. Gutiérrez Gallego and J. M. García
Fernández, Chem. Commun., 2004, 92 RSC .
210. J. L. Jiménez Blanco, P. Bootello, J. M. Benito, C. Ortiz Mellet and J. M. García Fernández, J.
Org. Chem., 2006, 71, 5136
211. Lipopolythioureas have been previously proposed as neutral DNA condensing agents for
systemic gene delivery. For selected examples see ref. 211–214: M. Breton, J. Leblond, J.
Seguin, P. Midoux, D. Shermann, J. Herscovici, C. Pichon and N. Mignet, J. Gene Med., 2010,
12, 45
212. J. Leblond, N. Mignet, C. Largeau, J. Seguin, D. Scherman and J. Herscovici, Bioconjugate
Chem., 2008, 19, 306
213. J. Leblond, N. Mignet, C. Largeau, M.-V. Spanedda, J. Seguin, D. Scherman and J.
Herscovici, Bioconjugate Chem., 2007, 18, 484
31
214. J. Leblond, N. Mignet, L. Leseurre, C. Largeau, M. Bessodes, D. Scherman and J.
Herscovici, Bioconjugate Chem., 2006, 17, 1200
215. A. Díaz-Moscoso, L. Le Gourriérec, M. Gómez-García, J. M. Benito, P. Balbuena, F. OrtegaCaballero, N. Guilloteau, C. Di Giorgio, P. Vierling, J. Defaye, C. Ortiz Mellet and J. M. García
Fernández, Chem.–Eur. J., 2009, 15, 12871
216. C. Byrne, F. Sallas, D. K. Rai, J. Ogier and R. Darcy, Org. Biomol. Chem., 2009, 7, 3763 RSC
217. A. Méndez-Ardoy, M. Gómez-García, C. Ortiz Mellet, N. Sevillano, M. D. Girón, R. Salto, F.
Santoyo-González and J. M. García Fernández, Org. Biomol. Chem., 2009, 7, 2681 RSC
218. A. Díaz-Moscoso, D. Vercauteren, J. Rejman, J. M. Benito, C. Ortiz Mellet, S. C. de Smedt
and J. M. García Fernández, J. Controlled Release, 2010, 146, 318
219. J. Rejman, A. Bragonzi and M. Conese, Mol. Ther., 2005, 12, 468
220. Incorporation of glycoligands in monodisperse polycationic amphiphilic CDs (polycationic
glyco-anphiphilic CDs; pGaCDs) has been recently proposed as a way to impart site-specific
gene delivery capabilities to such systems. See: N. Guilloteau, L. Le Gourrierec, A. DíazMoscoso, C. Ortiz Mellet, J. M. Benito, C. Di Giorgio, P. Vierling, J. Defaye and J. M. García
Fernández, Human Gene Ther., 2008, 19, 1157
221. H. Baigude and T. M. Rana, ChemBioChem, 2009, 10, 2449
222. M. S. Al-Dosari and X. Gao, AAPS J., 2009, 11, 671
32
Figure captions
Figure 1. Structure of some representative cationic polymers (top) and amphiphiles (bottom)
used for gene delivery. DOTAP: 2,3-dioleoyloxy trimethylammonium propane; DOPE: 2,3di(oleolyloxy)propyl
phospha-tidyl
ethanolamine;
DOSPA:
2,3-dioleoyloxy-N-[2(sperminecarboxiamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate; PLL: poly-Llysine; PEI: polyethyleneimine; PDMAEMA: poly(2-(dimethylamino)ethyl methacrylate);
chitosane: β(1→4)-linked glucosamine polymer .
Figure 2. General structure of cyclodextrins (CDs).
Figure 3. Structure of DIMEB (heptakis(2,6-di-O-methyl)-βCD; note that the commercially
available product is available as a mixture of regioisomers, the proportion of the homogeneous
derivative here represented varying from 50 to 95%) and of heptakis(6-deoxy-6-pyridylamino)βCD (1; the basic nitrogen atoms would be partially protonated under physiological
conditions).
Figure 4. Schematic representation of βCD-enhanced cholesterol-appended schizophyllan –
antisense ON complex formation.
Figure 5. Types of CD-containing polymeric systems.
Figure 6. Schematic representation of the structural diversity of CDPs investigated by Davis et
al.
Figure 7. Schematic structure of the folate -grafted βCD-PEI600 polymer highlighting each
component separately.
Figure 8. Schematic representation of pDNA compactation by CD-based supramolecular
cationic polymers described by Amiel et al.
Figure 9. Schematic representation of the elaboration of the transferrin-targeted pDNA- or
siRNA-CDP nanoparticles (RONDEL™) designed by Davis et al.
Figure 10. Illustration of the core–shell nanoparticles based on CD-grafted PEI–PBLA host –
guest interaction.
Figure 11. Structure of the pyrene-grafted βCD-pendant chitosane polymer described by Liu
and coworkers.
Figure 12. Schematic representation of αCD-based polyrotaxane–pDNA rotaCDplex formation.
Figure 13. In vitro time dependent transfection efficiency of the polyplexes formulated with
polyrotaxanes 15a and 15bversus bPEI-pDNA polyplexes in HEK 293 cells .
Figure 14. Structure of polypseudorotaxane 19 described by Liu et al.
Figure 15. Schematic representation of the structure and components of 2D βCD–
cucurbit[6]uril–PPG polypseudorotaxane 23 reported by Liu et al.
33
Figure 16. Schematic representation of CD-coated dendrimers (A) and CD-centered (starshaped) dendri(poly)mers (B).
Figure 17. Structure of ethylenediamine-based G0, G1 and G2 PAMAM dendrimers .
Figure 18. Structures of star-shaped βCD-centred cationic polymers reported by Xiao et al.
Figure 19. Relative orientation of the polycationic and hydrophilic domains in skirt-shaped (A)
and medusa-shaped (B) polycationic amphiphilic CDs (paCDs).
Figure 20. Structure of the dendritic paCD 50 (A) and in vitro gene expression efficiency (B,
bars) and cell viability (B, line) in BNL-CL2 cells of CDplexes obtained from paCDs 48, 49
(Scheme 7) and 50versus naked DNA and PEI-based polyplexes at N/P 5 (unfilled bars) and 10
(filled bars).
Figure 21. TEM micrograph of paCD 50:pDNA CDplexes with amplification of the ultrafine
structure of the particles and an schematic representation of the proposed arrangement of
paCDs and the DNA double helix (Reproduced with permission of Wiley-VCH from ref. 215).
Figure 22. Schematic representation of the structure of polycationic amphiphilic βCD click
clusters .
Figure 23. Schematic representation of the endocytic routes exploited by 60-pDNA complexes
for internalization and gene expression in Vero cells .
34
Figure 1
35
Figure 2
36
Figure 3
37
Figure 4
38
Figure 5
39
Figure 6
40
Figure 7
41
Figure 8
42
Figure 9
43
Figure 10
44
Figure 11
45
Figure 12
46
Figure 13
47
Figure 14
48
Figure 15
49
Figure 16
50
Figure 17
51
Figure 18
52
Figure 19
53
Figure 20
54
Figure 21
55
Figure 22
56
Figure 23
57
Scheme 1
Scheme 1 Synthesis of CD-containing cationic polymers (CDPs).
58
Scheme 2
Scheme 2 Synthesis of the βCD cationic click polymers described by Srinivasachari and
Reineke.
59
Scheme 3
Scheme 3 Schematic representation of the hydroxypropyl-βCD (HPCD)-PEI600 cationic
polymers reported by Wang and Yu. The synthesis involves the formation of carbamate bridges
by reaction of amino groups in bPEI and hydroxyl groups in HPCD with carbonyldiimidazole.
60
Scheme 4
Scheme 4 Synthesis of OEI-grafted βCD-PEG-PPG-PEG polyrotaxanes reported by Li et al.
61
Scheme 5
Scheme 5 Synthesis of αCD-PEG polyrotaxane 18.
62
Scheme 6
Scheme 6 Synthesis of CD-appended starburst G2 PAMAM dendrimers (also G3 and G4
PAMAM dendrimers were investigated).
63
Scheme 7
Scheme 7 Synthesis of α-mannosylated αCD-grafted PAMAM dendrimer 27 (a similar strategy
for α-galactosylated dendrimers was described).
64
Scheme 8
Scheme 8 Synthesis of βCD-grafted low generation PPI dendrimer 29.
65
Scheme 9
Scheme 9 Synthetic scheme for guanidinylated tetrapod 33.
66
Scheme 10
Scheme 10 Synthesis of αCD-centred OIE star polymers .
67
Scheme 11
Scheme 11 Synthesis of star-shaped cationic PDMAEMA-PEG copolymers based on βCD.
68
Scheme 12
Scheme 12 Synthesis of guanidino-CDs.
69
Scheme 13
Scheme 13 Synthesis of polycationic βCD click clusters .
70
Scheme 14
Scheme 14 Synthesis of thioalkylated polycationic amphiphilic CDs.
71
Scheme 15
Scheme 15 Synthesis of skirt-shaped cysteaminyl and thioureidocysteaminyl paCDs.
72
Scheme 16
Scheme 16 Synthesis of the medusa-shaped thioureidocysteaminyl paCD 56.
73
Scheme 17
Scheme 17 Synthesis of skirt-shaped paCD by photochemical thiol -allyl addition reported by
Darcy and coworkers.
74