Accepted Manuscript Title: Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI Author: Irene M. Mavridis Konstantina Yannakopoulou PII: DOI: Reference:

S0378-5173(15)00515-3 IJP 14956

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

31-3-2015 3-6-2015 4-6-2015

Please cite this article as: Mavridis, Irene M., Yannakopoulou, Konstantina, Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI.International Journal of Pharmaceutics This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI Irene M. Mavridis, Konstantina Yannakopoulou

Institute of Nanoscience and Nanotechnology, National Center for Scientific Research “Demokritos”, Agia Paraskevi Attikis, P.O.Box 60037, 15310 Greece.

Email: Irene M. Mavridis – [email protected] Telephone : 0030 2106503793

Abbreviations used in this article: α-, β-, γ-CD, α-, β-, γ-cyclodextrin; SBEβCD, sulfobutylether-β-CD; CM-βCD, carboxymethyl β-CD; CE, capillary electrophoresis; EDTA, ethylenediaminetetraacetic acid


Abstract The review presents a full library of single-isomer primary rim per-carboxylate- and per-sulfate-α-, -β- and –γ-cyclodextrin (CD) derivatives and their potential for pharmaceutical nanotechnology. Recent advances in cyclodextrin chemistry have enabled robust methods for the synthesis of single-isomer anionic CDs. Numerous nanobio-applications have been already reported for these negatively charged derivatives, which alone or in combination with other biodegradable molecular platforms can become important carriers for targeted drug delivery and release. Specialized applications are also discussed, such as chiral separations, as well as the ability of per-6-carboxylated-cyclodextrins to coordinate with metal cations and especially with lanthanide cations that makes them candidates as contrast agents for Magnetic Resonance Imaging.

Keywords: Anionic cyclodextrins; negatively charged; carboxylate-; sulfate-; drug carriers; cation binding; Chemical compounds studied in this article: beta-CYCLODEXTRIN (PubChem CID: 444041); alpha-CYCLODEXTRIN (PubChem CID: 444913); gammaCyclodextrin (PubChem CID: 86575); Sugammadex (PubChem CID: 6918584); Hydroxypropyl-beta-cyclodextrin (PubChem CID: 90479739)


1. Introduction Cyclodextrins (CDs) are carbohydrate oligomers obtained by enzymatic degradation of starch. The most common comprise six, seven and eight α-1,4linked-D-glucopyranose residues (α-, β-, γ-CD, respectively). CDs have the shape of hollow truncated cones (D'Souza et al., 1998; Dodziuk, 2006) forming an internal relatively hydrophobic cavity (Scheme 1), whereas their exterior is hydrophilic, lined by numerous hydroxyl groups: 6, 7, 8 primary OH groups on the narrow rim and 12, 14, 16 secondary OH groups on the wider rim of α-, β-, γ-CD, respectively. They are water soluble and able to include in their cavity a plethora of molecules of small and medium molecular weight, even parts of biological macromolecules (Stella et al., 1999). Due to their non-toxic nature (Stella and He, 2008) and their low immunogenicity (Davis and Brewster, 2004), CDs have been used in pharmaceutical technology as excipients of lipophilic drugs (Davis and Brewster, 2004; Kurkov and Loftsson, 2013; Loftsson and Duchêne, 2007 ) in order to increase aqueous solubility and bioavailability, as well as improve the stability of drugs (Loftsson and Brewster, 1996). CDs are also used in analytical separations and in food and cosmetic technology (Szejtli, 1998, 2004). The mechanism leading to these favorable properties is primarily molecular encapsulation, although recent studies have proposed that inclusion is only a part of more complex phenomena involving also external interactions among the components and aggregation, phenomena which cannot be readily quantified (Kurkov and Loftsson, 2013; Loftsson and Brewster, 2013).


Because of availability in large quantities and thus low cost, β-CD has been the most commonly used member of the family, in spite of its lower aqueous solubility (1.8% w/v) compared to α-CD (14.5% w/v) and γ-CD (23.2% w/v). Presently, several drugs with formulations involving natural CDs are found in the markets of Japan, Europe and USA (Loftsson and Duchêne, 2007 ). By chemical modification of the CD hydroxyl groups, further synthetic CDs can be produced and indeed numerous such macrocycles have been synthesized that extend the potential application repertoire towards nanobiotechnology, e.g. cell internalization and gene delivery (Mourtzis et al., 2008; Srinivasachari et al., 2008; Yannakopoulou, 2012), nanomaterials for drug delivery (Zhang and Ma, 2013) or environmental applications (Badruddoza et al., 2010).

Scheme 1 Most of the earlier chemical modifications were performed on the most abundant β-CD, primarily aiming at increasing its solubility in water. Depending on their chemical nature, the introduction of substituents improves or impairs the inclusion complexation ability for guest molecules (Kraus, 2011; Yannakopoulou, 2012). Simple methylation has procuced versatile derivatives with flexible cavities and improved solubility profiles, such as the randomly methylated-β-CD (~50% w/v solubility). The derivatives sulfobutyl ether-β-CD (captisol®) and 2-hydroxypropyl-


β-CD (HPβCD) are two notable examples of approved excipients with solubility in water exceeding 60% and 50% w/v, respectively. Moreover, they possess superior solubilizing ability (up to 5000-fold enhancement of drug solubility in water) ( and additionally they exhibit better toxicity profiles (Arima et al., 2011; Stella and He, 2008) that makes them more suitable for pharmaceutical applications. The above modified CDs are approved excipients for six marketed products (Kurkov and Loftsson, 2013). An advantage of modified cyclodextrins (Kahn et al., 1998) is that they can encapsulate larger guests due to elongation of their cavity. Compared to natural CDs, anionic derivatives collectively form stronger complexes with polar or oppositely charged drug molecules (Adam et al., 2002; Maffeo et al., 2006). A remarkable case is sugammadex, an anionic γ-CD derivative, that forms an exceedingly strong inclusion complex with rocuronium bromide (vide infra, 4.2, 5.1) (Adam et al., 2002) and has been approved by the European Medicines Agency, not as an excipient but as a drug (commercial name: bridion), to be used in neuromuscular block reversal following general anesthesia. Further, the ability of anionic CDs to discriminate between the enantiomers of drugs (Cherkaoui and Veuthey, 2002; Vaccher et al., 2006; Zhu and Vigh, 2003) is used extensively for the separation of the latter. Anionic CD derivatives have been also tested in vitro as artificial enzymes (e.g. glycosyl phosphorylases) (Rousseau et al., 2005) and in antiviral studies (Grammen et al., 2014; Leydet et al., 1998), as well as for transdermal delivery of nonpolar drugs by iontophoresis (Juluri and Murthy, 2014)


and for biomedical applications such as for trypsin inhibition for development of oral delivery of peptides and proteins (Ooya et al., 2002). Further, anionic per-6carboxylate-CDs have enhanced chelating potential for metal cations (e.g. of calcium, iron and copper) (Chatain et al., 2004; Trotta et al., 2001) and for the preparation of Ag nanoclusters, which exhibit strong antimicrobial ability in vitro (Wang et al., 2013). Their most remarkable property is the binding of lanthanide cations that makes them potential contrast agents for Magnetic Resonance Imaging (MRI) (Kotková et al., 2012; Maffeo et al., 2010) one of the most powerful diagnostic methods. The above examples suggest that future nano-bioapplications of anionic CD derivatives hold great promises, thus current research explores both synthesis of smart derivatives and diverse areas of possible applications. Presently, anionic cyclodextrins as vehicles for biomedical applications is reviewed for the first time. A library of chemically homogeneous CDs of defined composition and structure (single-isomers), prepared up to now is presented. Moreover, the methods of synthesis are outlined, because it is important that such highly pure CDs are used in pharmaceutical nanotechnology applications. Thus per-6-carboxylate and -sulfate anionic CDs are reported, since single-isomer per6-phosphorylate CDs modified either in the primary or in the secondary sites have not been synthesized up to now (Agostoni et al., 2015; Grachev, 2013). The article additionally reviews self-assembly of anionic CDs, geometry of complexes, drug inclusion and release, as well as enantiomeric discrimination. Finally, exploration


of potential applications of anionic CDs is discussed and progress for their applications in pharmaceutical nanotechnology is presented.

2. Library of single-isomer per(6-substituted) anionic cyclodextrins Anionic CD derivatives have been synthesized early and several are used successfully (e.g. sulfobutyl ether-β-CD), however they are not single-isomers, but randomly substituted mixtures of regioisomers. Single-isomer derivatives have been synthesized mostly after 2000, because cyclodextrin chemistry has matured considerably since then. Scalable and reproducible synthetic and purification procedures to obtain pure single-isomer anionic CDs is very crucial in order to perform the binding, pharmacokinetic and toxicity studies of drug/CD complexes (Loftsson et al., 2015) on defined CD derivatives. Only then such studies will allow the advance from the proof-of-concept stage to the clinical tests and eventually the market. In this part, synthesis, properties and binding ability of highly symmetrical single isomer, anionic per(6-substituted)-CDs are examined.

2.1. Per(6-carboxylate)-CD derivatives In general, per-6-substituted anionic CDs comprise a subgroup of what is called Janus cyclodextrins, because they have distinctly different primary and secondary faces. The first single isomer synthesis, per(6-O-carboxylate)-CD, appeared in 1997 (Kraus et al., 1997). Cyclodextrins bearing mixed acidic and basic moieties are also included in the list. These have been synthesized in the


hope that they would show stronger and more selective complexation and solubilization of guests compared to the natural CDs. Moreover, they were expected to exhibit strong binding at a specific pH range and weaker at another, properties that would render them more suitable for drug binding and release. 2.1.1. Derivatives with a single carboxyl group on each α-D-glucopyranose unit Although direct oxidation of the CD primary hydroxyl groups does not result in per(6-carboxylate)-CDs, nevertheless these derivatives (1 and 2 in Scheme 2) are included, since they are the simplest per(carboxylate)-CDs with a single carboxyl group on each α-D-glucopyranose unit. Initially reported direct oxidation of natural α- and β-CDs to carboxyl groups (Fraschini and Vignon, 2000) has yielded a mixture of regioisomers of carboxylated hosts. In contrast, per(2,3-di-Omethyl)- and per(2,3-di-O-acetyl)-α- and β-CDs under similar oxidation conditions, comprising TEMPO/sodium hypochlorite, have been smoothly converted into the corresponding per(5-carboxy-5-dehydro)-CD derivatives 1a-d (Kraus et al., 2000). Quantitative hydrolysis of 1c-d afforded the homogeneous derivatives per(5carboxy-5-dehydro)-α- and β-CDs (2a and 2b, respectively), with the carboxyl groups directly attached to C5 (Scheme 2).


Scheme 2 Carboxymethylation of the hydroxyl groups of parent β-CD to obtain 4 (Scheme 2) by reaction with chloroacetic acid in alkaline conditions (NaOH) could not be cleanly implemented (the products were mixtures of positional isomers) (Reuben et al., 1994; Tan et al., 2012), unless the secondary hydroxyl groups were methylated. Specifically, per-6-O-carboxymethylation took place efficiently only if the very reactive ethyl diazoacetate was used as the alkylating agent resulting exclusively in 3a-b, α- and β-CD derivatives, respectively (Kraus et al., 1997). Compounds 1a and 1b, being amphiphilic, are reported to form strong head-tohead dimers (vide infra 3.0) in organic solvents (Kraus et al., 2000). The carboxymethylated β-CD (4) (Scheme 2) was only recently obtained from heptakis(2,3-di-O-allyl-6-t-butyldimethylsilyl)-β-CD in a rather involved synthetic sequence and together with 3b were studied as MRI contrast agents (vide infra 5.2) (Idriss et al., 2013).


Substitution of halogens of per(6-deoxy-6-bromo- or -iodo-α-, -β- and - γCDs by carboxyl group-bearing thiol or phenol nucleophiles has afforded a large collection of CD derivatives 5a-e (Cameron et al., 2002) and 6a-e, respectively (Scheme 3). Derivatives 6a-e were prepared from the reaction of the corresponding octakis(6-triflate)-γ-CD with an appropriate carboxylated phenol (Cameron et al., 2002). Similar β-CD derivatives (5f-g, Scheme 3) have been synthesized by the same procedure (NaH/DMF) (Steffen et al., 2007). The γ-CD derivatives 5a-5e and 6a-6e have been found to act as artificial hosts for rocuronium bromide, a steroidal neuromuscular blocker, of which 5a(m=2) displayed the optimum binding profile (vide infra 4.2) (Adam et al., 2002).

Scheme 3 2.1.2. Derivatives with two or more carboxyl groups on each glucopyranose unit


Heptakis(6-O-dicarboxy)-β-CD 7b (Scheme 4) has been prepared (Trotta et al., 2001) from the unsaturated precursor heptakis(6-maleate)-β-CD, 7a (Ding et al., 2010; Huang et al., 2011; Trotta et al., 2001) by Michael addition of thioacetic acid to the double bond. The products 7a and 7b exhibit great solubility in water and ability to complex with many divalent metallic cations, e.g. they act as sequestrants for calcium cations.

Scheme 4 Further, using the unsaturated per(2,3,6-O-allyl)-α-, β- and γ-CDs, 7c-e (prepared from the parent CDs using allyl iodide in the presence of sodium hydride) the polycarboxylates 8a-c and 9a-c were obtained possessing 18, 21, and 24 and 36, 42, and 48 carboxylate groups, respectively (Scheme 4) (Leydet et al., 1998; Ni et al., 2002). Synthesis of polycarboxylates 8a-c was effected from 7c-e by 11

photochemical addition of 3-mercaptopropanoic acid and a catalytic amount of a,a’-azobis(isobutyronitrile). Derivatives 9a-c were synthesized under the same conditions using 2-mercaptosuccinic acid. Derivatives 8 and 9 were tested for inhibition on the replication of two different strains of HIV-1 (vide infra 5.1) (Leydet et al., 1998). 2.1.3. Derivatives bearing both amino and carboxyl groups on each glucopyranose unit The above mentioned per-allylated CDs 7c-e (Scheme 4) have been used as intermediates to produce polyzwitterionic derivatives 10a-c (Scheme 5) by radical addition of triethyl boron-protected cysteine via formation of a diethylboroxazolidone complex with its amino and carboxylic functions (Leydet et al., 1998). Derivatives 10a-c they have been tested for inhibition on the replication of HIV-1 as above (2.1.2). Acidic and basic functionalities on the primary face of β-CD have been introduced by direct attachment of amino acids as the appended groups (Ashton et al., 1996). Reaction of cysteine with heptakis(6-iodo-6-deoxy)-β-CD (in Na/liq. NH3 at -33 ºC) afforded heptakis(6-cysteinyl-6-deoxy)-β-CD (11a, Scheme 5). On the other hand, N-Boc-cysteine reacted smoothly with heptakis(2,3-dimethyl-6bromo-6-deoxy)-β-CD under simple alkaline conditions (KOH/DMSO), while deprotection of the amino group afforded heptakis(2,3-di-O-methyl-6-cysteinyl)-βCD (11b). Both derivatives are highly water-soluble.


Scheme 5 In the effort to improve selectivity and efficiency of binding with specific guests via Coulomb and π-π stacking interactions, derivatives 12a,b (Scheme 6) have been synthesized (Kraus et al., 1998; Roehri-Stoeckel et al., 1997). Dipolar cycloaddition of the dimethyl butynedicarboxylate on either heptakis(2,3-di-Oacetyl-6-azido-6-deoxy)-β-CD or heptakis(2,3-di-O-methyl-6-azido-6-deoxy)-β-CD and subsequent ester hydrolysis afforded the corresponding dicarboxy-1,2,3triazolyl-2,3-derivatives 12a and 12b. Further studies on their complexation properties have not been reported. Synthesis of strong binders of metal ions has been achieved by the introduction of iminodiacetic acid [bis(carboxymethyl)amino groups] on all primary hydroxyl sites of CDs, resulting in EDTA-type bifurcated structures of the appended substituents in derivatives 13a-13c (Scheme 6). Thus, per(2,3-di-Omethyl-6-amino-6-deoxy)-α-, β- and γ-CDs reacted with t-butyl bromoacetate to a


afford, after subsequent deprotection in trifluoroacetic acid, compounds 13a-c (Maffeo et al., 2010) that have the ability to coordinate strongly with lanthanide ions (vide infra 5.2.1).

Scheme 6 Polycarboxylate CDs have also been synthesized by attaching the polydentate ligands DTTA (diethylenetriaminetetraacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) on the narrow rim (Scheme 7). DTTA is an analogue of DTPA (diethylenetriaminepentaacetic acid). Both DTPA and DOTA are currently used in the clinic as MRI contrast agents. A novel alkyne-functionalized DTTA has been clicked on heptakis(6-azido-6-deoxy)β-CD to produce monodisperse derivative 14 (Bryson et al., 2008). Similarly, the DOTA analogues 15 of α- and β-CD have been prepared (Kotková et al., 2012) from per(6-amino-6-deoxy)-α- and β-CDs and the isothiocyanate derivative of a DOTA phosphinic acid. The DTTA and DOTA polycarboxylated moieties hang from the primary rim of cyclodextrins in derivatives 14 and 15 as molecular arms and bind to lanthanides with favorable properties (vide infra 5.2.1).


Scheme 7 2.1.4 Derivatives with oligomers /polymers on each glucopyranose unit Grafting of polymeric or oligomeric moieties on the primary rim of cyclodextrins have been also attempted in order to produce new conjugate materials enriched with the capabilities of the CD macrocycles, especially as nanocarriers for drug release. Thus the single-isomer derivative 16 was derived by the reaction of the star-shaped poly(γ-benzyl-L-glutamate) with octakis(6-amino-6deoxy)-γ-CD followed by hydrolysis of the benzyl ester (Yong et al., 2013) (Scheme 8). The metal binding ability of 16 is analyzed in 5.1.


Scheme 8 Further, the β-cyclodextrin dendrimer 17a (Scheme 8) has been synthesized (Newkome et al., 1998) by coupling heptakis(6-amino-6-deoxy)-β-CD with the isocyanide precursor of the t-butyl ester of the appropriate tricarboxylate dendron followed by formic acid hydrolysis of the intermediate. Coupling of 17a with Behera’s amine (the corresponding amine of the isocyanide precursor) and subsequent hydrolysis of the obtained t-butyl ester gave 17b. Both carboxylated dendrimers 17a and 17b retain their inclusion complexation and recognition properties and they can be employed as polyfunctional monomers for selfassembly. Further properties of these dendrimers have not been reported so far. More complex carboxylated derivatives have been synthesized that contain acidic and basic moieties and indeed exhibit very strong binding constants with guests bearing acidic or basic groups under a specific pH range and weaker at another, a desirable property that renders them most suitable for drug release (vide


infra 5.1). Specifically, the per(6-folic acid) appended α-, β- and γ-CDs 19a-c having one caproic acid spacer between folic acid (FA) and the CD macrocycles (Scheme 9) (Okamatsu et al., 2013a) and the corresponding 19d derivative of βCD with two caproic acid units as spacer (Scheme 9) (Okamatsu et al., 2013b) have been synthesized. The synthesis has initiated from per(6-amino-6-deoxy)-α, β- and γ-CDs, which were condensed with Boc-caproic acid (Boc-6aminohexanoic




methylmorpholinium chloride (DMT-MM), followed by deprotection, which resulted in the fully per-substituted-6-amino-caproic acid-CDs 18a-c (Scheme 9). For the spacer with two moieties of caproic acid, 18d, the latter reactions were repeated using the β-CD derivative 18b as the starting CD. Then folic acid derivatives were obtained by condensation of 18a-d and FA via its γ-carboxyl group using DMT-MM to afford the desired modified CDs 19a-d.

Scheme 9



Per(6-sulfate)-CD derivatives The first single-isomer sulfomethylether-β-cyclodextrin derivative was

synthesized in 1997 (Vincent et al., 1997b). However, randomly substituted sulfoalkylether-cyclodextrins, i.e. mixtures of isomers, randomly substituted at carbon-positions 6, 2 and 3 of the glucopyranose units and characterized by an average degree of substitution were first synthesized in 1990 (Rajewski, 1990) and have been used in studies for drug delivery and enantioselectivity, as well as for other purposes e.g. inhibitors for the viability of Plasmodium falciparum (malaria) in human and murine Plasmodium cultures (Crandall et al., 2007). Sulfobutyl etherβ-cyclodextrin (SBE-β-CD) is the most successful such derivative, a nonnephrotoxic polyanionic β-CD (Luke et al., 2010; Stella and He, 2008), which is marketed under the name captisol® by Janssen. SBE-β-CD has been approved by Food and Drug Administration (FDA) as an excipient. At least four active pharmaceutical ingredients use SBE-β-CD as a drug carrier (Kurkov and Loftsson, 2013; Stella and He, 2008). The simplest members of the series of single-isomer per(6-sulfate)-α-, -βand -γ-CDs, 20a-c (Scheme 10) have been synthesized from the corresponding per(2,3-di-O-acetyl)-CD analogues 21a-c, respectively by deacetylation (Li and Vigh, 2004a; Vincent et al., 1997a; Zhu and Vigh, 2003). Compounds 21a-c were obtained from removal of (t-butyldimethyl)silyl (TBDMS) groups of intermediates per(2,3-di-O-acetyl-6-O-TBDMS)-CDs, which subsequently were sulfated with the pyridine.sulfur trioxide complex in DMF (Li and Vigh, 2003; Vincent et al., 1997b;


Zhu and Vigh, 2000). The per(2,3-di-O-methyl-6-sulfate)-CDs 22a-c, (Scheme 10) were likewise synthesized from the corresponding per(2,3-di-O-methyl-6-OTBDMS)-CDs (Busby et al., 2003; Cai et al., 1998; Li and Vigh, 2004b) by deprotection at the primary side and subsequent sulfation with the pyridine.sulfur trioxide complex in DMF, as above.

Scheme 10 Three more heptakis(6-sulfate)-β-CDs, 23, 24 and 25 with nonidentical substituents at the C2, C3, and C6 positions have been prepared, adopting a synthetic approach of general applicability (Scheme 11). In summary, the synthesis of derivative 23 (Busby and Vigh, 2005b) started from the intermediate heptakis(6-O-TBDMS)-β-CD (βCD-6-O-TBDMS). The C2 hydroxyl groups were then protected with triethylchlorosilane to obtain heptakis(2-O-triethylsilyl-6-OTBDMS)-β-CD, which was methylated with iodomethane/sodium hydride yielding heptakis(2-O-methyl-3-O-triethylsilyl-6-O-TBDMS)-β-CD. Next, the triethylsilyl protecting group was selectively removed yielding the intermediate heptakis(2-Omethyl-6-O-TBDMS)-β-CD (also useful for derivatives 24 and 25). The C3 hydroxy groups of the latter were acetylated, the primary side groups were subsequently


deprotected and finally were sulfated using the pyridine.sulfur trioxide complex in DMF to afford derivative 23. Heptakis(6-O-TBDMS)-β-CD has been converted with similar methods to derivatives 24 (Busby and Vigh, 2005a) and 25 (Maynard and Vigh, 2000) (Scheme 11).

Scheme 11. Synthetic methodology for synthesis of single-isomer, heptakis(6sulfate)-β-cyclodextrins carrying nonidentical substituents at the C2, C3 positions. Amphiphilic sulfated cyclodextrins have also been synthesized by addition of hexanoyl group in the secondary site of the CDs (Dubes et al., 2003), which are


analogues of 22a-c, i.e. per(6-sulfate-2,3-di-O-hexanoyl)-α-, -β- and -γ-CDs (26ac) by protection-deptotection methods similar to the above. Finally, two more single isomer heptakis(6-sulfate)-β-CDs (Scheme 12) have been synthesized, heptakis(2,3-di-O-methyl-6-O-sulfopropyl)-β-CD, 27, (Kirschner



2005; Takeo

et al.,




sulfonatophenyloxy-6-deoxy)-β-CD, 28, (Schwinté et al., 1999). The former has been prepared as its analogue compound 22b and the latter, bearing aromatic sulfate appendage, has been synthesized by reaction of 4-hydroxyphenylsulfonic acid disodium salt with heptakis(6-bromo-6-deoxy)-β-CD.

Scheme 12

3. Self-assembly of anionic CDs The amphiphilic derivatives 1a-b of α- and -β-CDs (Scheme 2), form headto-head homodimers in 1,2-dichloroethane and in chloroform, by multiple hydrogen bonding via the carboxylic groups in the narrow rim (Figure 1, dimerization mode a) (Kraus et al., 2000). This property is due to the propensity of the carboxylic


groups to form dimers, which in this case is facilitated by the rigidity of the CD skeleton. Such behavior has not been reported for per-carboxylate derivatives 35, probably due to the flexibility in the conformations of the spacer groups intervening between the CD rim and the carboxyl end-groups, because of the repulsive forces among the latter. In contrast, heterodimers are reported (Figure 1, dimerization modes b) between corresponding anionic (heptakis(6-Scarboxylate)-β-CD, 5f) with cationic (heptakis(6-amino)-β-CD) in water (Hamelin et al., 1995), which shows that assembly is taking place, despite the high dielectric constant of the solvent. The latter association has been confirmed by 1H NMR, potentiometric and theoretical studies (Hamelin et al., 1998; Jullien et al., 1999; Schwinté et al., 1999) and more recently by isothermal titration and NMR studies for the pairs heptakis(6-carboxylate)-β-CD 5f (or a partially phosphorylated)-β-CD) and heptakis(6-guanidino)-β-CDs (Fotiadou et al., 2011).

Figure 1. Head-to-head dimers (a) via H-bonding of the carboxylic groups; (b) via H-bonding and Coulomb forces. Color code: red, negatively charged group, blue, positively charged group.


4. Inclusion complexes of anionic CDs

4.1 Binding ability High binding constants of drug/CD complexes are important

for the

enhanced bioavailability of the drug in water, although it is required that binding and pharmacokinetic studies are repeated in physiological conditions (in the presence of plasma proteins) before proceeding to the use of the macrocycle in any formulation (Leong et al., 2015a; Loftsson et al., 2015). Intuitively, it is expected that the binding constants of inclusion complexes of charged guests would be higher for oppositely charged CD hosts, because of mutual Coulomb interactions, which would be further strengthened by the hydrophobic host-guest interactions in the CD cavity. As early as 1978 (Matsui and Okimoto, 1978), binding studies between a positively charged CD and a negatively charged guest showed that the binding constant at pH 10.5 is higher than that with β-CD by a factor of 4. Comparative studies (Okimoto et al., 1996) on binding of the sulfated SBE-β-CD and neutral HP-β-CD with seven drugs in their neutral and charged forms showed that the binding constants of the neutral and cationic forms of the five drugs with SBE-β-CD are higher compared to the HP-β-CD, whereas for the anionic forms they were similar. The differences for the cationic forms of the drugs were significantly superior to these of the neutral forms. Further, isothermal titration calorimetric studies (Wenz et al., 2008) of anionic, cationic and neutral guests with an identical skeleton (p-substituted


derivatives of t-butylbenzene) with heptakis-6-substituted-β-CD derivatives (cationic, anionic, and neutral, respectively) show that the binding constants of the complexes range from 10 X 106 to 381 X 106 M-1 and depend on the total free binding energy. It is suggested that the latter has an apolar and an electrostatic contribution, which can be calculated using Coulomb’s law, if the geometry of the inclusion complex is known and an accurate assumption of the charges can be made. Earlier calorimetric and NMR studies in aqueous solutions had also examined the nature of the binding interactions and arrived to similar conclusions using the positively charged per(6-amino-6-deoxy)-α- and -β-CDs and the negatively charged β-CD 5f (Scheme 3) with oppositely charged guests, as compared to the native CDs (Kano et al., 2000). The studies propose that the two contributions to the free binding energy, enthalpic gain and entropic gain, derive from (a) Coulomb and van der Waals interactions enhancing stabilization of inclusion cooperatively (enthalpic gain) and (b) entropic gain is promoted by dehydration of both the hosts and guests. The above reasoning of enthalpic and entropic gain has been applied for the binding mechanism of heptakis(6-Ocarboxylate)β-CD and metal-coordinated guests, e.g. M(phen)3n+ (where M is Ru(II) or Rh(III) and phen is 1,10-phenanthroline) and its interaction with DNA (Kano and Hasegawa, 2001).


Geometry of inclusion


It has been shown that per-carboxylate-β-CDs, such as 5f and 5g are very suitable for binding polar guests, with which they align in antiparallel fashion (Wenz et al., 2008). Indeed antiparallel alignment of the host and guest dipoles is exhibited in numerous inclusion complexes, especially in these involving Coulomb host-guest interactions. A very characteristic example of the above is the inclusion complex of octakis[6-deoxy-6-(3-sulfanylpropionic acid)]-γ-CD (Scheme 3, 5a, m=2) with the positively charged steroidal neuromuscular blocker rocuronium bromide (Adam et al., 2002; Bom et al., 2002). In this inclusion complex, the tertiary amine of the drug is located on the primary side surrounded by the host’s carboxylic groups (Figure 2) and this electrostatic interaction contributes to the observed strong binding of the complex, which has the highest binding constant, ≥107 M-1, observed in CDs (Cameron et al., 2002). The selection of the derivative 5a (m=2) for the capture of rocuronium bromide resulted from systematic screening (Adam et al., 2002; Cameron et al., 2002) of the series of synthesized single-isomer per(6-carboxylate)-CDs 5a-e and 6a-e. The strong binding of 5a (m=2) is favored both enthalpically and entropically (see 4.1). Besides the electrostatic interactions, enthalpic gain results also from van der Waals contacts with the extended lipophilic cavity that encapsulates all the steroidal rings (Figure 2) and the thio-linkers of the substituents contributing considerably, in contrast to the oxygen-linker analogues 6a-e. Entropic gain comes from dehydration of the host and the drug as in other systems discussed in 4.1.






representation of the structure of the rocuronium bromide/octakis[6-deoxy-6-(3sulfanylpropionic acid)]-γ-CD (5a, m=2) complex.

Antiparallel binding has also been observed in the complex of the same host 5a (m=2) with penicillin drugs, in which protection of the drugs against β-lactamase is suggested by deceleration of hydrolysis of the β-lactam ring (Maffeo et al., 2006). Specifically, 2D ROESY NMR spectra in buffered aqueous solution (pH 7) suggested inclusion of ampicillin and amoxicillin with their carboxyl group away from the sulfanylpropionic side of 5a. The presented systematic studies of binding of a specific guest to several macrocycles-receptors are very tedious due to the effort required to synthesize the hosts. Thus methods are being developed for computational screening of derivatized cyclodextrin hosts with high affinity and selectivity for specific drugs in order to facilitate their design, a process inverse to the structure-based drug


design. Such an example is the screening of a virtual library of heptakis-substituted β-CDs (positively or negatively charged) and their corresponding monosubstituted analogues in order to select the optimum host for inclusion of the antineoplastic agent camptothecin (Steffen et al., 2007).


Discrimination between drug enantiomers Efficient separation of enantiomers is very significant in pharmaceutical

research because information on enantiomeric purity of optically active drugs is required prior to their marketing. As chiral hosts, cyclodextrins are able to resolve enantiomeric guests due to formation of diastereomeric inclusion complexes (Cucinotta et al., 2010; Leong et al., 2015b). Anionic cyclodextrins have been examined for enhanced enantiomeric discrimination of chiral guests (Zhang et al., 2013), such as drugs (Servais et al., 2005), amino acids (Kitae et al., 1998) and peptides (Kano et al., 2001) or for development of methods for enantiomeric purity determination (Mikuma et al., 2015; Orlandini et al., 2015; Rousseau et al., 2010). Capillary electrophoresis (CE) and chromatography are the preferred methods for such studies, however other analytical methods can be used (alone or in combination with CE), such as NMR spectroscopy and Mass spectrometry (MS) or optical spectroscopy (Zhou et al., 2003). Thus NMR studies of methyl esters of three dipeptides (Kano et al., 2001) showing the trend Ala-Ala-OMe < Ala-LeuOMe < Ala-Trp-OMe for binding stability with the anionic CD 5f (Scheme 3) suggests van der Waals host-guest interactions of the carboxy-terminal amino acid and Coulomb interactions between the carboxyls of the host and the charged 27

amino-terminal of the dipeptide. Higher enantioselectivity of 5f for the (R,R) as compared to the (S,S) enantiomers, is explained as due to the better fit of the R enantiomer of the carboxy-terminal amino acid in the CD cavity. Up to now, the consensus is that no general scheme of the enantiomeric discrimination of CDs has resulted from such comparative studies i.e. how to design cyclodextrin modifications in order to improve effectiveness for resolution of enantiomers. As it is also shown in the following paragraph, this is closely related to the nature and geometry of the specific host-guest interactions. The latter conclusion has been verified in systematic studies using singleisomer α-, β- and γ- cyclodextrin derivatives 20-25 (vide supra 2.2), all completely sulfated in the C6-positions, several of them additionally substituted on their larger secondary rims with moderately hydrophobic (acetyl) and/or hydrophobic (methyl) functional groups (Schemes 10 and 11), which have been examined for enantiomeric discrimination of drugs and related pharmaceutical compounds by capillary electrophoresis (Busby et al., 2003; Busby and Vigh, 2005a, b; Cai et al., 1998; Li and Vigh, 2003, 2004a, b; Maynard and Vigh, 2000; Vincent et al., 1997a; Vincent et al., 1997b; Zhu and Vigh, 2000, 2003). The use of pure single-isomers in these studies has been stressed out as an absolutely mandatory condition for two reasons: (a) to eliminate the drawback of several isomers in commercially available sulfate CDs resulting in ill-defined complex mixtures and therefore properties and (b) to provide a comprehensive theoretical framework of the migration of the enantiomers in electrophoresis and identify the variables in order to predict successful separations. A common screening kit comprising groups of


structurally diverse nonionic, strong base, weak base, weak acid and zwitterionic enantiomers of drugs (analytes) have been used [72 of them are listed in (Busby et al., 2003)] for the evaluation of the resolving ability of the above anionic CDs. Under this scheme, 20b (heptakis(6-sulfate)-β-cyclodextrin) (Vincent et al., 1997a) can be broadly applicable for chiral resolution in CE forming stronger complexes with most compounds of the screening kit than the moderately hydrophobic hosts 21b (heptakis(2,3-di-O-acetyl-6-sulfate)-β-CD) (Vincent et al., 1997b) and the more hydrophobic 22b (heptakis(2,3-di-O-methyl-6-sulfate)-β-CD) (Li and Vigh, 2004b). However, these three sulfated hosts have different separation selectivity towards the tested analytes and could be used as complementary chiral resolving agents having specific applications in separations of enantiomers. Different binding constants and separation selectivity towards many of the analytes of the screening kit were also observed among the γ-CD analogues 20c, 21c and 22c, as well as among the corresponding α-CD analogues 20a, 21a and 22a. Moreover, the separation selectivity among the α-CD and γ-CD analogues was often complementary. Thus the above single-isomer sulfated CDs can have specific applications in CE separations of enantiomers, because rapid and efficient enantiomer separations have been observed for a large number of structurally diverse analytes, under various conditions and pHs. The general trend is that the efficiency decreases from the β-CD, to γ-CD, to α-CD analogues. The binding strengths and separation selectivity towards this set of analytes observed for the hosts 23-25 (Scheme 11) did not exhibit substantial improvement among hosts 2022 other than that they were often different. In summary, this study shows that lack


of information of host-guest intermolecular interactions at the molecular level does not permit up to now to understand and explain differences in enantioselective efficiency, thus methods allowing for structure determination, as NMR, MS and Xray crystallography, are clearly needed in order to escape from the trial-and-error mode of screening (Busby and Vigh, 2005b). Nevertheless, it has been shown that single-isomer CD derivatives facilitate the development of separation methods for samples of multiple components (Culha et al., 2000; Feng et al., 2015) and that by simple, accurate and sensitive modifications CE methods (Zhou et al., 2002), have determine








pharmaceutical interest using the single-isomers 20b, 21b, 22b and 21c along with randomly substituted CD analogues. 5. Anionic CD derivatives in pharmaceutical nanotechnology Natural and modified CDs have been coupled to other drug carrier platforms to enhance and improve their function. CDs have the advantage that they act in a dual fashion: (a) they endow the resulting nanoassemblies with the capacity to carry selectively drugs into their cavity and (b) they can be functionalized by various moieties (groups or whole molecules, such as peptides, proteins, antibodies) in order to target specific cells. In this respect, anionic CDs have been conjugated to dendrimers (Saraswathy et al., 2015), magnetic nanoparticles (Ding et al., 2015) and mostly to polymers (Cevher et al., 2014; Hamilton and Breslin, 2014). Most of the reported applications are in conjunction with chitosan (CS), a, hydrophilic, non-toxic and biocompatible cationic polysaccharide (composed of β(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine randomly distributed)


derived from partially de-acetylated chitin. The natural mucoadhesive properties of CS (Grabovac et al., 2005) and its ability to open the tight junctions among epithelial cells are improved/enhanced by association with CDs, e.g. acquiring ability to carry hydrophobic drugs (Kono and Teshirogi, 2015; Venter et al., 2006). Thus two common randomly substituted anionic CDs, carboxymethyl β-CD (CM-βCD) and sulfobutylether-β-CD (SBE-βCD) are conjugated to the positively charged CS (Fülöp et al., 2015) to form nanoparticles (NPs) by ionotropic gelification [a NP forming method for hydrophilic polymers (Calvo et al., 1997)]. These NPs are less toxic than the naked CS NPs and can perform gene delivery in Calu-3 cells, a model for human airway epithelial cells (Teijeiro-Osorio et al., 2009) under conditions that adequately represent the in vivo situation. The anionic CDs have a stabilizing effect on the NPs preventing aggregation in simulated intestinal medium, thus they are of particular interest for oral drug delivery (Anraku et al., 2015; Chen et al., 2008; Trapani et al., 2008). NPs of CS and SBE-βCD were used for the delivery and release of econazole nitrate to ocular mucosa in a model study for ocular delivery (Mahmoud et al., 2011), as well as of the peptide glutathione (Trapani et al., 2010). NPs of CS and CM-βCD have been used to study the delivery and release of ketoprofen (Prabaharan and Mano, 2005) and the macromolecules insulin and heparin (Krauland and Alonso, 2007). Two typical examples are (a) CM-βCD coupled with glycol chitosan (an ethylene glycol derivative of chitosan), which interacts with porcine gastric mucin and is a pH sensitive carrier of the anticancer drug doxorubicin (since it forms a strong inclusion complex at pH 7.4 and a week one at pH 5.0 (a pH resembling the


environment of cancer cells) thus doxorubicin is released) (Tan et al., 2012); (b) SBE-βCD/CS nanoparticles loaded with ciprofloxacin have been used to coat Titanium surfaces and are shown to be promising for microbial protection of Titanium implants (Mattioli-Belmonte et al., 2014). 5.1.

Applications of single-isomer anionic CDs The above new composite materials based on anionic cyclodextrins,

randomly substituted without defined composition, have not reached clinical verification, as other CD-polymer conjugates (Davis, 2009; Davis et al., 2010). However, the reported results show clearly that anionic CDs have important potential in pharmaceutical nanotechnology for advanced targeted delivery and controlled release of drugs and further experimental work may lead to clinical trials as single-isomer CD derivatives become more abundant and commonly used. As already mentioned, the most successful case of a single-isomer anionic per(6substituted)-CDs is 5a, (m=2) that forms an extremely stable inclusion complex with the neuromuscular blocker rocuronium bromide (Adam et al., 2002; Bom et al., 2002) and it has been approved as a drug (trade name Bridion) for administration to patients after general anesthesia, since the encapsulation of rocuronium bromide into the CD cavity, reverses completely its effects and promotes quicker recovery. The following cases range from simple drug inclusion in single-isomer anionic CD derivatives for bioavailability enhancement or enantiodiscrimination to more complex applications that involve supramolecular assemblies or conjugates.


The single-isomer heptakis(6-maleate)-β-CD, 7a (Scheme 4) (Trotta et al., 2001), which is very soluble in water has been used for solubilization of two drugs (both forming 1:1 inclusion complexes), 4-acetaminophenol (K a = 1.43 X 103 M-1) (Ding et al., 2010), a well-known drug for the treatment of pain and fever and mefenamic acid (MA), a non-steroidal anti-inflammatory drug with antipyretic and strong analgesic properties (Ka = 7.15x102 M-1) (Huang et al., 2011). The solubility of both drugs is greatly enhanced and the thermal stability of the former improved. For the latter drug, a simple and practical spectrofluorimetric method has been developed for determination of MA in bulk aqueous solution and other media based on the drug’s enhanced (more than double) fluorescence intensity in the presence of 7a (detection limit, 3.36×10−9 mol L-1 and detection range 9.00 × 10−5 to 2.00 × 10−8 mol L-1). The method has been used to detect MA in tablets, urine and serum. Related to the above is the use of the heptakis(6-sulfate)-CD 21b (Scheme 10) for the detection of amphetamine and five more related compounds in urine samples. The analysis of the enantiomers of the six compounds was performed simultaneously by a mass spectrometry-capillary electrophoresis method (Iio et al., 2005). The single-isomer amphiphilic sulfate per(6-sulfate-2,3-di-O-hexanoyl)-α-, β- and -γ-CDs 26a-c that combine the antiviral activity of sulfated CDs with their inclusion ability (Dubes et al., 2003), have been used for the binding of acyclovir, a guanosine analog (9-[(2-hydroxyethoxy)methyl]guanine), an essential antiviral drug. The α-, β-CD derivatives 26a and 26b form 1:1 inclusion complexes with the drug, which resides in the secondary side region of the hosts occupied by the long


hexanoyl chains, whereas the γ-CD analogue exhibits 1:2 host:guest stoichiometry, the same as the corresponding complexes with the natural α-, βand γ-CD analogues. In the latter cases, the second occupation site is suggested to be the cavity. The stability constants range from 895 mol L-1 for 26a/acyclovir to 1036 mol L-1 for 26b/acyclovir to 1647 mol L-1 for a partially substituted (only four sulfate groups) analogue of 26c/acyclovir. A related application is the use of single-isomer polyanionic cyclodextrins as anti HIV agents. Sulfated polymers, exhibit antiviral activity, however it appears that polyanions with a well-defined structure, as cyclodextrins (Leydet et al., 1997) and high number of anionic groups are more effective against HIV, because it has been shown that they interfere between the envelop glycoprotein of the virus and the receptors in the host cells. Thus numerous sodium salts of the poly carboxylated α-, β- and γ-CDs 8a-c, 9a-c (Scheme 4) and 10a-c (Scheme 5) have been tested (Leydet et al., 1998) for their inhibition on the replication of two different human immunodeficiency virus types, HIV-1 (strains IIIB and HE), HIV-2 (strains of ROD and EHO) and simian immunodeficiency virus SIV (MAC251) in MT-4 cells. It has been shown that all compounds inhibit the HIV-1 strain IIIB (at concentrations, IC50, of 0.1-2.9 μM) (and the HE at 4-fold higher concentrations). The activity of 8a-c is comparable to the CD sulfates with 14 and 16 anionic groups (Leydet et al., 1997). All compounds 8-9 showed activity against SIV MAC251 virus and some of them against strains ROD and EHO of HIV-2, but at higher concentrations. The zwitterionic analogues 10a-c were not active against HIV.


A single-isomer polymer-cyclodextrin system is derivative 16 (Scheme 8) involving appendage of seven poly(L-glutamic acid) chains on the primary face of β-cyclodextrin (Yong et al., 2013). This system has been used to develop nanoparticles (micelles) for the targeted delivery of the anti-cancer drug cisdichlorodiammine platinum (II) (CDDP). As shown in Figure 3, the cis-platinum drug forms coordination complexes (16/CDDP) with the carboxyl groups of 16. Subsequently, micelles were produced through inclusion of the adamantyl group of adamantyl-derivatized polyethylene glycol in the CD cavities of the 16/CDDP conjugate. The latter exhibited sustained drug release of CDDP over 50 hours in PBS and decreased cytotoxicity to KB cells compared to cis-platinum alone.

Figure 3. Schematic process for the assembly of the adamantyl-mPEG-16/CDDP micelles. The single-isomer per(6-folic acid-appended)-CDs 19a-d via caproic acid linkers have been evaluated as carriers for antitumor drugs. Analogues of α-, β-


and γ-CDs were linked with tumor targeting folic acid molecules (FA) via one caproic acid (19a-c, Scheme 9) (Okamatsu et al., 2013a) or for the β-CD analogue 19d via two caproic acid molecules (Okamatsu et al., 2013b). These ionisable derivatives (mixed anionic and cationic moieties) forming strong complexes with doxorubicin (DOX) under a specific pH value and weaker in another, in combination with the targeting properties of FA (Su et al., 2014) can develop into appropriate drug carriers. The strongest antitumor activity is exhibited by 19b (Figure 4). The Ka value of DOX/19b complex is markedly high (1.7 × 106 M−1) at pH 7.3, i.e. the pH of the bloodstream and decreases (5.4 × 104 M−1) at pH 6.8, the pH value in the Folate Receptor(FR)-α-mediated endosome. It is reported that a Ka value of more than 104 − 105 M−1 is required to maintain a stable complex in vivo (Stella et al., 1999), thus the DOX/19b complex has desirable K a characteristics to associate in bloodstream and to dissociate in endosomes after cellular uptake through FR-α-mediated endocytosis.


Figure 4. Schematic structure of the heptakis(6-folic acid-caproic acid-appended)β-CD. Interaction of CDs with porphyrins is of special interest due to the importance of the latter as photosensitizers (capable of producing various reactive oxygen species) in Photodynamic Therapy (PDT) for cancer and atherosclerosis and Photodynamic Antimicrobial Chemotherapy (PACT). Inclusion of porphyrins into the CD cavity changes their aqueous solubility and photophysical properties and reduces aggregation, therefore CDs are used in PDT and PACT studies. Anionic CDs 20b and 22b [heptakis(6-sulfate)-β-CD and heptakis(2,3-di-O-methyl6-sulfate)-β-CD, respectively] have been reported in a PACT study (Mosinger et al., 2009), in which several porphyrins have been used (Scheme 13) in order to determine if the excited states of the photosensitizers are influenced by inclusion in the CD cavity The positively charged photosensitizer TMPyP has high affinity to form inclusion host-guest complexes with the negatively charged CDs 20b and 22b, due to Coulomb interactions. This results in increased basicity of the TMPyP’s triplet states causing protonation even in neutral solution. In contrast, the same


photosensitiser interacts with the external surface of native β-CD and does not form inclusion complexes, thus its triplet states are not affected. On the other hand, β-CD forms 1:1 host-guest complexes with the diprotonated porphyrin TPPSH22+, as well as with the nonprotonated TPPS, therefore the inclusion complex formation increases the lifetimes of the photosensitisers’ triplet states.

Scheme 13 It is well known that CDs have been used as models for enzyme catalysts (Das et al., 2008). The inclusion complex of the anionic carboxymethyl β-CD 3b (Scheme 2) with porphyrin TGPP (Scheme 13) has been used as the core of a supramolecular system that models artificial anionic receptors of cytochrome c (cyt-c) (Kano and Ishida, 2007). Many attempts have been made to devise artificial receptors with negatively charged surfaces, which can be recognized by cyt-c through electrostatic interactions, since it is known that exposed positively charged


lysine residues on its exterior interact with negatively charged residues of its partners, cyt c reductase and oxidase. Based on the strong ability of per(2,3,6-triO-methyl)-β-CD (TMe-β-CD) (which is identical to 3b in the secondary side) to include peripheral aryl groups of water-soluble meso-tetraarylporphyrins (Kano et al., 2005), 3b and TGPP were used to form the ternary 2:1 complex 3b/TGPP (K = 2 X 105 M-1), with two exposed polyanionic surfaces formed by the seven anionic carboxymethyl groups in the primary sides of β-CDs. This 2:1 complex binds electrostatically two cyt-c molecules (Figure. 5). Fluorescence quenching of TGPP upon addition of cyt-c in the 3b/TGPP complex suggests proximity of cyt-c to TGPP, albeit corresponding only to two thirds of the TGPP molecules, which indicates that in one third of cyt-c molecules the hemin centers are away from TGPP. The geometry of the supramolecular complex 3b/TGPP/cyt-c interaction shown in Figure 5 has been verified by using the neutral TMe-β-CD (it forms 2:1 host:guest TMe-β-CD/TGPP complexes) in place of 3b. In this case no fluorescence quenching of TGPP occurs upon addition of cyt-c due to the inability of the TMe-β-CD/TGPP complex to bind cyt-c, because of the absence of electrostatic interactions. The advantage of the above design is its supramolecular


multimodular nature, which can be easily optimized by changing certain modules, in contrast to classical synthetic receptors formed covalently.

Figure 5. A suggested schematic structure of the complex 3b/TGPP/Cytc. 5.2.

Metal cation binding of single-isomer anionic CD derivatives As it has been mentioned before, poly-carboxylated CDs display metal ion

binding properties (Idriss et al., 2013; Trotta et al., 2001), which are very important for development of materials and methods for biomedical and environmental applications. Thus anionic CDs bearing randomly substituted carboxymethyl groups (CM-β-CD) have been used as remediation agents to remove metal cation pollutants from mixed-contaminated soils, e.g. CM-β-CD has been used very efficiently for extracting arsenic, copper, iron and 2,3,4,6-tetrachlorophenol from soil (Chatain et al., 2004), as well as to modify Fe3O4 magnetic nanoparticles capable to absorb and desorb Cu2+ ions at different pH values (Badruddoza et al., 2010; Badruddoza et al., 2011). CM-β-CDs and sulfated cyclodextrins and their complexes with lanthanides Dy(III) or Yb(III) have been proved very effective in enantiomeric


discrimination of various drugs in 1H NMR spectra, because they cause shifts much larger than parent CDs (Wenzel et al., 2003). Further, due to the antibacterial activity of Ag nanoparticles (Ag-NPs) against Gram-positive and Gram-negative bacteria (Prabhu and Poulose, 2012), CM-β-CD has been used as stabilizing and reducing agent of naked Ag-NPs for the preparation of Ag nanoclusters, which exhibit strong antimicrobial ability in vitro (Wang et al., 2013). 5.2.1. Single-isomer anionic CD derivatives as contrast agents for MRI It is the binding with lanthanide cations what makes carboxylated CDs very important, because they can serve as contrast agents for MRI, the most powerful diagnostic method for cancer, cardiovascular and musculoskeletal disease. Anionic CDs are different from other contrast agents in that they can (a) carry a drug in the cavity in addition to binding the lanthanide cations; (b) be further derivatized with targeting moieties for specific tissues. Thus biomolecular imaging can be coupled with transporting a drug and targeting the cells to which the latter should be transported. Moreover, CDs are excellent host scaffolds, because their toxicity is negligible. Contrast agents (CAs) are used to enhance the image contrast between body soft tissues. The majority are complexes of the paramagnetic cation Gd(III). A measure of CA efficiency is relaxivity r1, i.e. enhancement of relaxation rate of water protons due to the CA, normalized to 1 mM solution of the agent. Relaxivity depends on the rate of exchange of the water molecule(s) coordinated to the paramagnetic Gd(III) ion of the CA, as well as on the rotational correlation time of


the entire molecule. Therefore molecules bound to many Gd(III) cations and of high molecular weight would show improved contrast properties. In recent years, the quest for CAs efficient at higher magnetic fields is very important due to modern research MRI instrumentation shifted to magnetic fields higher than the 1.5 Tesla scanners routinely used today. Lanthanide(III) cations interact preferentially with polyanionic ligands and specifically carboxylates to form strong complexes (Beeby et al., 2000; Li and Selvin, 1995) owing to a large electrostatic contribution to the binding. The following paragraphs report on cyclodextrin derivatives, compounds of intermediate molecular mass, i.e. less than 10 kDa, whose relaxivities are very high therefore they are potential contrast agents. The anionic CDs 13a-c (Maffeo et al., 2010) (Scheme 6) that carry iminodiacetic acid groups, feature the ethylenediaminetetraacetic acid (EDTA) chelation ability to form strong complexes with Eu(III), Tb(III), and Gd(III) ions. Specifically, the derivatives form multimetal complexes, as 13c binds exactly four metal ions (Figure 6), 13b binds mainly three and 13a two to three metal ions (Maffeo et al., 2010). Additionally, the presence of ~1.5 exchangeable water molecules coordinated to each metal cation has been revealed by luminescence lifetime measurements. Further, 1H NMR measurements of molecular relaxivity for the Gd(III) complexes of 13a-c afforded values of molecular relaxivity higher than those








polycarboxylate commercial contrast agents: 4 to 10 times at 0.28 Tesla (12 MHz), and 6 to 20 times at 2.3 Tesla (100 MHz), reaching a maximum of 92.9 (± 9.3) mM1

s-1 that corresponds to 23.2 mM-1 s-1 per Gd(III) cation. The latter value was


largely retained in human blood plasma [50.2 (± 0.9) mM-1 s -1, or 6.3 mM-1 s-1 per Gd(III) cation] indicating sufficient stability at 25 C for 2.5 h. MTT tests of the Gd(III) complexes in human skin fibroblasts did not show toxicity. The above lanthanide complexes with the described advantageous properties are attractive for application as versatile molecular imaging probes.

Figure 6: Structure of the octakis[6-bis(carboxymethyl)amino-6-deoxy-2,3-Omethyl]-γ-cyclodextrin - Gd(III) complex, as calculated by PM3 semiempirical quantum mechanical calculations (Maffeo et al. 2010). Color code: For atoms, grey, carbon; red, oxygen; blue, nitrogen; violet, gadolinium(III). For water molecules: cyan. Using a different strategy, the clinically used compounds DTPA and DOTA have been attached onto the primary rim of CDs as molecular arms. Thus


lanthanide complexes of the monodispersed cyclodextrin derivatives 14, 15 (Scheme 7) exhibit very improved properties as compared to DTPA and DOTA. The β-CD derivative with DTPA binds seven Gd(III) (14.Gd(III)) or Eu(III) cations (Scheme 14), each cation associating with an average of 1.8 exchangeable water molecules (Bryson et al., 2008). This metal cluster yielded high relaxivity properties [43.4 mM-1 s -1 per molecule corresponding to 6.2 mM-1 s-1 per Gd(III) cation at 9.4 Tesla (400 MHz)] and enhanced contrast using a human MRI scanner compared to DTPA (3.2 mM-1 s-1 at 9.4 Tesla).

Scheme 14 Similarly, DOTA analogues of α- and β-CD clusters 15, coordinate six and seven Gd(III) and Eu(III) cations via their arms, respectively (Kotková et al., 2012). The Gd(III) conjugate complex 15.Gd(III) of the β-CD analogue showed a high molecular relaxivity at high fields, reaching ~140 mM-1 s-1 at 1.5 Tesla (64 MHz)


and 100 mM-1 s -1 at 3 Tesla (128 MHz) at 25 ºC, (20 and 14.2 mM-1 s-1 per Gd(III) cation, for the respective magnetic fields). 6. Conclusion Anionic cyclodextrins exhibit improved properties for drug inclusion compared to natural CDs, e.g. they have higher aqueous solubility and longer cavity that allowing encapsulation of larger size drugs, they form stronger inclusion complexes with polar guests or guests bearing positive charge at a given pH, thus potentially allowing for pH regulated drug release. Two anionic derivatives have already entered the market, (a) SBE-βCD (sulfate-β-CD), which is an excipient in at least four products approved by FDA, (b) the single-isomer sugammadex [octakis(6-O-carboxylate)-γ-CD], which is a drug itself for the elimination of the effect of anesthesia after surgery. Moreover, extensive studies show the potential of single-isomer anionic CDs for biomedical applications, e.g. as antiviral agents, drug carriers, artificial receptors, as well as of the effectiveness of single-isomer per(6-sulfate)-CDs for chiral separations of drugs and other compounds of pharmaceutical interest. A significant property of per(6-O-carboxylate)-CDs is the binding of metal ions. Well-designed carboxylated CDs are able to coordinate lanthanide cations and they have been demonstrated to act as excellent contrast agents for MRI, especially suitable for high magnetic fields, which will be the future environment of this clinical method. In addition, the presence of numerous carboxylate or sulfate groups facilitates further functionalization of the CD hosts by attachment of targeting or labeling (e.g. fluorophore) groups, thus enabling achievement of additional characteristics, i.e. to act as advanced drug carriers 45

beyond solubilization and stabilization of drugs. Moreover, anionic CDs have been coupled with other biodegradable molecular platforms endowing them with the wide biocompatibility, negligible toxicity and the other favorable characteristics of CDs in order to be applied to biomedical research as composite multifunctional drug nanocarriers. As the range of applications of anionic CDs is expanding through laboratory exploration, it is anticipated that ultimately more derivatives will find use in the pharmaceutical industry and nanotechnology. Towards this target, the availability of pure isomers of the anionic derivatives will be crucial, since it will allow the full and detailed safety, toxicity and biocompatibility examinations, which are prerequisite for any further exploitation and use, either as individual macrocycles or as domains in larger assemblies. Therefore reproducible and scalable methods for the synthesis of single-isomer derivatives is of strategic importance for advancement in the field. Akwnoledgements We acknowledge the Marie Curie Programs No. 237962 CYCLON (FP7-PEOPLEITN-2008) and No. 608407 CycloN-Hit (FP7-PEOPLE-ITN-2013) for financial support.

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Figure legends

Figure 1. Head-to-head dimers via (a) H-bonding between the carboxyl groups; (b) H-bonding and Coulomb forces. Color code: red, negatively charged group; blue, positively charged group.

Figure 2. A schematic representation of the structure of the rocuronium bromide/octakis[6-deoxy-6-(3-sulfanylpropionic acid)]-γ-CD (5a, m=2) complex.

Figure 3. Schematic representation of the assembly of adamantyl-mPEG with the Pt-complex of carboxylated βCD (16/CDDP) to form micelles.

Figure 4. Schematic structure of the heptakis(6-folic acid-caproic acidappended)-β-CD.

Figure 5. Proposed structure of the complex 3b/TGPP/Cyt-c.

Figure 6: Structure of the octakis[6-bis(carboxymethyl)amino-6-deoxy-2,3-Omethyl]-γ-cyclodextrin - Gd(III) complex, as calculated by PM3 semiempirical quantum mechanical calculations (Maffeo et al. 2010). Color code: For atoms, grey, carbon; red, oxygen; blue, nitrogen; violet, gadolinium(III). For water molecules: cyan.


Scheme legends Scheme 1. Structures of α-, β-, γ-CDs. Carbon atom numbering is shown for a single D-glucopyranose unit. Scheme 2. Structures of the single-isomer CD derivatives 1-4 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 3. Structures of the single-isomer CD derivatives 5-6 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 4. Structures of the single-isomer CD derivatives 7-9 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 5. Structures of the single-isomer CD derivatives 10-11 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 6. Structures of the single-isomer CD derivatives 12-13 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 7. Structures of the single-isomer CD derivatives 14-15 as described in the text (n = 6, 7 correspond to α-, β-CD, respectively). Scheme 8. Structures of the single-isomer CD derivatives 16-17 as described in the text (n = 7, 8 correspond to β-or γ-CD, respectively). Scheme 9. Structures of the single-isomer CD derivatives 18-19 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 10. Structures of the single-isomer CD derivatives 20-22 as described in the text (n = 6, 7, 8 correspond to α-, β-or γ-CD, respectively). Scheme 11. Synthetic methodology for synthesis of single-isomer, heptakis(6-


sulfate)-β-cyclodextrins carrying nonidentical substituents at the C2, C3 positions. Scheme 12. Structures of the single-isomer β-CD derivatives 27-28 as described in the text. Scheme 13. Structures of neutral (TGPP) and positively (TMPyP) or negatively charged (TPPS, TPPSH22+) porphyrins. Scheme 14. Structures of the single-isomer β-CD derivatives 14 and 15 as coordination complexes with gadolinium-III cations.


Graphical Abstract


Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI.

The review presents a full library of single-isomer primary rim per-carboxylate- and per-sulfate-α-, -β- and -γ-cyclodextrin (CD) derivatives and thei...
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