Laboratory of Structural & Supramolecular Chemistry

Research

B. Synthesis of functionalcyclodextrin derivatives and applications

  1. Modified CDs display novel properties

Synthetic modifications of cyclodextrins result in introduction of functional groups that endow the host molecules with specific properties [1-3] ( Fig. B1), allow elongation of the host cavity to capture long guest molecules or the combination of the above that permits direct inclusion in specific direction [4,5] (Fig. B2).

Fig. B1. Synthesis of mono-aminobenzoic acid-substituted β-cyclodextrins and supramolecular self inclusion in the crystalline state [1].

Fig. B2. Per(carboxyethylthio)-β-cyclodextrin encapsulates the elongated dye methyl orange (left) in a sense opposite to that of  natural β-cyclodextrin (right) [5].

  1. Positively charged CDs for bio-applications

Per-6-modified CDs with guanidino or lysine- or arginine-like groups (Fig. B3) comprises a very interesting family of hosts that have the ability  to (i) penetrate cell membranes, (ii) transport molecules (iii) compact DNA and therefore perform transfection [6,7]. These properties have been used for transfection of green fluorescent protein (GFP) (Fig. B4) .

Fig. B3. Positively charged CDs

Fig B4. Guanidino and amino cyclodextrins: (left) penetrate cell membranes (fluorescent microscopy image of HeLa cells) and (right) perform DNA transfection that expresses GFP protein (green fluorescence) .

In addition, the above positively charged β- and γ-cyclodextrin derivatives effectively inhibited anthrax toxin action by blocking the transmembrane oligomeric pores formed by the protective antigen (PA63) subunit of the toxin (Fig. B5), whereas α-cyclodextrins were ineffective [8, 9] in collaboration with V. Karginov, Innovative Biologics, USA.

Fig. B5. Positively charged β- and γ-cyclodextrin: A Bacillus anthracis toxin pore blocker

III. Negatively charded CDs with a range of applications

EDTA-type CDs, i.e. CDs modified with aminodiacetyl groups have been synthesized, and characterized (Fig. B6) [10]. Their main property is the ability to coordinate with metal ions, especially lanthanides and particularly gadolinium, Gd(III). The EDTA-CDs coordinate with Gd(III) and fast exchanging water molecules to form metal clusters that display high relaxivity values, especially at high (100 MHz) magnetic fields. Gd-EDTA-CDs exhibit significantly higher relaxivity characteristics compared to existing MRI contrast agents

Fig. B6.  GEDTA (EDTA-γ-cyclodextrin) (center )and its Gd(III) complex bearing four Gd(III) ions (right) as calculated by PM3 semiempirical methods [10]

  1. Glycoclusters

Attachment of recognition sugars in the primary side of cyclodextrins results in formation of glycoclusters (Fig. B7) able to attach on bacterial membranes, thus having possible biomedical applications [11].

Fig. B7. A representative cyclodextrin-based glycocluster with the possibility to recognize cell surface lectins

  1. Surface active CDs

A stable, well-packed undecenyl-cyclodextrin (DMBUA) monolayer on Si/SiO2/ novolac resin (AZ) substrate (Fig.  B8) is able to detects triclosan from a 10 nM aqueous solution in a reflectance spectrometer [12].

Fig. B8. Representation of a stable CD monolayer able to detect the antibacterial triclosan (shown schematically).

An elementary supramolecular conducting system was constructed using a novel (±)-thioctic acid-functionalized βcyclodextrin host deposited on a gold (Au) surface and an iridium-bearing guest molecule with biphenyl tails to insert specifically into the cyclodextrin cavity (Fig. B9). The resulting Au surface functionalised by  this supramolecular system was used to investigate remote electron communication between Au and the Pt/Ir tip of a Scanning Tunneling Microscope. I-V spectroscopic analysis of the tunneling current through the supramolecular layer revealed the relation between the effective height of the barrier and tunneling distance [ 13].

Fig. B9. Representation of the supramolecular system conducting current between the Au substate and the Pt/Ir STM tip.

  1. Cyclodextrin derivatives for production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNOS)

CD derivatives covalently linked with photoactive units provide new systems for molecular transport that combine the inclusion properties of the CDs with photo-triggered properties of the attached unit (multimodal systems). These systems can be employed as photosensitizers (PS) in Photodynamic Therapy (PDT)  for selectively treating neoplastic lesions: PS is accumulated in the tumor and is activated by light of the appropriate wavelength in order to generate reactive oxygen species (ROS), which are highly deleterious to cells.

Most of the PSs used in PDT are porphyrins. The covalent attachment of a CD moiety to a pophyrin can be envisaged as a strategy to combine the drug enacapsulation/solubilization capacity of the CD with the photosensitizing/fluorescence imaging properties of the porphyrin, thus creating a multimodal drug carrier. As an immediate consequence, CD-porphyrin conjugation could enhance the aqueous solubility of the porphyrin and promote its de-aggregation as well as increased fluorescence intensity and lifetime.  The ultimate test of these systems would be their ability to become internalised by cells and preferably display a localisation preference into a certain subcellular compartment.

Thus we have synthesised a porphyrin-βCD conjugate exhibiting the typical red fluorescence of the porphyrin  that encapsulates a nitric oxide photodonor tailor-made to fit the βCD cavity (Fig. B10), which aggregates into a nano-assembly of 16 nm (a supramolecular bichromo­phoric aggregate).  This system retains porphyrin fluorescence thus enabling its imaging into living cells and is able to generate nitric oxide and singlet oxygen under illumination by visible light. It has been proven to be internalized in melanoma cells and induce a significant level of mortality [14], probably due to the combined action of RNOS and ROS , in collaboration with S. Sortino, U of Catania, It.

Fig. B10. A supramolecular nanoaggregate able to produce light-triggered ROS and RNOS

An approach to use protoporphyrin IX (PpIX) for effective application of PDT in cancerous lesions is the topical or systemic administration of 5-aminolevulinic acid (ALA) and its esters, which results in increased production and accumulation of PpIX. The use of  ALA for PpIX biosynthesis is attractive, but has twoshortcomings: large concentrations of exogenous ALA (in order to bypass the negative feedback control exerted by heme on enzymatic biosynthesis of PpIX from ALA) and the strong dimerization propensity of ALA.  To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, βcyclodextrin (βCD) was implemented [15]. The resulting PpIXβCD has both carboxylic termini of PpIX connected to the CD (Fig. B11). PpIXβCD is water soluble, that has been found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines, while its phototoxiciy is comparable to that of PpIX. Moreover, PpIXβCD effectively solubilized the breast cancer drug tamoxifen metabolite Ndesmethyltamoxifen (NDTAM) in water. Thus PpIX-βCD is a bimodal βCD derivative and its PpIXβCD/NDTAM complex has been readily internalized by both cell lines employed (Fig. B11).

Fig. B11. The multimodal action of PpIX-βCD. Phototoxic effect of PpIX and transport of tamoxifen metabolite N-desmethyltamoxifen by βCD.

On another research line, the grafting of SNO groups on the cyclodextrins can afford reactive nitric oxide species (RNOS) generating carriers upon photochemical or thermal stimulation (Fig. B12). SNO-βCDs were characterized in detail for the first time regarding conformational preferences and SNO group content, thermal and photochemical stability, ability to en­capsulate guest molecules as well as cell toxicity and cell permeation[16]. The CD cavity is available for guest encapsulation without noticeable perturbation of the -SNO functionality while hosting e.g. the chemotherapeutic tamoxifen. Nitrosation of per-SH-βCD to form per-SNO-βCD was found to compete with SNO decomposition and disulfide bridge formation, resulting in an average 5.2 SNO groups instead of 7. Mono-SNO-βCD is water soluble, whereas multi-SNO-βCD is DMSO soluble. Both have satisfactory thermal stability, cell permeability and they were found to be chemically non toxic to cells at considerably high incubation concentrations (>200 μM). Thus the combination of RNOS-generating-hosts with RNOS-designed-guest may provide powerful phototoxic systems for PDT applications.

Fig. B12. Left: synthesis of SNO-CDs. Right: bimodal action of a RNOS host carrying a guest molecule

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References