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

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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.

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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 viral-vector-based gene delivery therapeutic due to these concerns.

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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

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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 βCD-containing 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.

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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 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-6-pyridylamino)-β-cyclodextrin 155 (Fig. 3, right) embedded into multilayered films (e.g. poly-L-lysine/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 cholesterol-appended derivatives of schizophyllan , a fungal polysaccharide consisting of a β(1→3)-D-glucan 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

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(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 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

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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 1,3-dipolar 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

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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 adamantane-modified 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™ (RNAi-oligonucleotide 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-tosyl-βCD with the commercial polymers . Though toxicity was alleviated, CD grafting was detrimental for transfection efficiency as compared to naked PEI. However, co-formulation

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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

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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-poly-ε-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-polypropyleneglycol-polyethyleneglycol (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 2,4,6-

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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

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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 anthranyl-functionalized 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

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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

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concern.153,154 To circumvent this drawback, Uekama and coworkers exploited the bio-compatibilizing 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 CD-PAMAM 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 α-galactosides164 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.

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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 6-monoisothiocyanate-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 14-mer 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 by-

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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 self-organize 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

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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 self-assembly 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.

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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 mercaptopropionic esters to per-2-O-allylated βCD derivatives, has been reported by Darcy and coworkers (Scheme 17).216

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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 growing understanding of the CD-based gene delivery mechanisms, it is likely that several others will follow.

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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.

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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: 2,3-di(oleolyloxy)propyl phospha-tidyl ethanolamine; DOSPA: 2,3-dioleoyloxy-N-[2-(sperminecarboxiamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate; PLL: poly-L-lysine; 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.

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Figure 16. Schematic representation of CD-coated dendrimers (A) and CD-centered (star-shaped) 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 .

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Scheme 1

Scheme 1 Synthesis of CD-containing cationic polymers (CDPs).

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Scheme 2

Scheme 2 Synthesis of the βCD cationic click polymers described by Srinivasachari and Reineke.

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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.

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Scheme 4

Scheme 4 Synthesis of OEI-grafted βCD-PEG-PPG-PEG polyrotaxanes reported by Li et al.

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Scheme 5

Scheme 5 Synthesis of αCD-PEG polyrotaxane 18.

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Scheme 6

Scheme 6 Synthesis of CD-appended starburst G2 PAMAM dendrimers (also G3 and G4 PAMAM dendrimers were investigated).

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Scheme 7

Scheme 7 Synthesis of α-mannosylated αCD-grafted PAMAM dendrimer 27 (a similar strategy for α-galactosylated dendrimers was described).

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Scheme 8

Scheme 8 Synthesis of βCD-grafted low generation PPI dendrimer 29.

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Scheme 9

Scheme 9 Synthetic scheme for guanidinylated tetrapod 33.

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Scheme 10

Scheme 10 Synthesis of αCD-centred OIE star polymers .

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Scheme 11

Scheme 11 Synthesis of star-shaped cationic PDMAEMA-PEG copolymers based on βCD.

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Scheme 12

Scheme 12 Synthesis of guanidino-CDs.

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Scheme 13

Scheme 13 Synthesis of polycationic βCD click clusters .

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Scheme 14

Scheme 14 Synthesis of thioalkylated polycationic amphiphilic CDs.

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Scheme 15

Scheme 15 Synthesis of skirt-shaped cysteaminyl and thioureidocysteaminyl paCDs.

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Scheme 16

Scheme 16 Synthesis of the medusa-shaped thioureidocysteaminyl paCD 56.

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Scheme 17

Scheme 17 Synthesis of skirt-shaped paCD by photochemical thiol -allyl addition reported by Darcy and coworkers.

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