Structural & Supramolecular Chemistry Research Group – Research – Cyclodextrin Chemistry

A. Cyclodextrin Chemistry: designed derivatives for bio- and nano-bio- applications

The laboratory studies supramolecular systems comprising mainly cyclodextrins (CDs), which are water soluble macrocyclic carbohydrates of varying size (α-, β-, γ-CD) possessing a hydrophobic cavity (Figure 1A). In this cavity a plethora of hydrophobic molecules can be encapsulated. The numerous hydroxyl groups that line the narrow (primary) side and the wide (secondary) side of the macrocycles are responsible for the aqueous solubility, structural stability and versatility in functionalization of the macrocycles.

Figure A1: (a) Side view of the cyclodextrin structure depicting the glucopyranose repeating unit, n=number of units in the cycle. (b) Top view of the α-, β-, and γ-CD macrocycles in a 3D CPK representation; color code: red =oxygen, light grey=hydrogen, dark grey=carbon; the hydrophobic  character of the cavity  is due to the lining with H–C- and –O– atoms. (c) Top view of the chemical structure of β-CD.

Synthetic modifications of cyclodextrins with selected functional groups allow extension of the host cavity to capture longer guest molecules but also endow the host CDs with tailored properties: inclusion of specific selected guests, preferential/directed orientation of insertion, multi-fold enhanced binding and increased aqueous solubility. The modified CDs are primarily used for drug delivery. Attachment of photoactive molecules on the cyclodextrin scaffold result in versatile multimodal molecular nanocarriers. Examples are:

A1. Positively charged cyclodextrins (PCCDs) clickable

α-, β- and γ-CD derivatives bearing lysine- or arginine-like groups (Figure A2) comprise a very interesting family of hostsA1 that have the ability to penetrate cell membranes, transport molecules and DNA intracellularly and act as transfection agents (Figure A3).A1-3 The cell internalization process follows to a considerable extend an endocytosis pathway. Additionally, several of these derivatives are able to block the channels of pore forming proteins found in many pathogenic bacteria (such as anthrax) and thus inhibit infection, due to their positive charge, symmetry and size suitability (in collaboration with Dr. V. Karginov, USA).A4 Recently an optimally designed PCCD was shown to bind strongly with the antibiotic oxacillin and protect it from specific oxa-1 β-lactamase (from an E-coli strain) hydrolysis by ~60% (in collaboration with Dr. V. Miriagou, Hellenic Pasteur Institute).A5

Figure A2: Positively charged cyclodextrin derivatives


Figure A3. Positively charged cyclodextrins (PCCDs) cross cell membranes and transfer their cargo along: (a) fluorescent-labeled PCCDs show intracellular localization in murine macrophages (green spotted areas); (b) intracellularly transfered DNA via a PCCD expresses the Green Fluorescent Protein (right image) in HEK 293T cells.

Recent studies have also revealed that PCCDs enable phosphorylated gemcitabine (the bioactive form of the potent anticancer drug gemcitabine, a nucleoside drug) to penetrate efficiently into aggressive human breast cancer cells (MCF7), eventually leading to a substantial reduction of IC50 values. Internalization of phosphorylated gemcitabine is not favored due to high hydrophilicity and instability of the molecule therefore the help of PCCD nanocarriers is critical. Moreover, compared to the free drug, phosphorylated gemcitabine encapsulated in PCCDs displayed substantially improved in vitro activities also on the aggressive human cancer cells CCRF-CEM Ara-C/8 C, a nucleoside transporter-deficient T leukemia cell line. The current study offers the proof-of-concept that active phosphorylated nucleoside drugs could be efficiently transported by PCCDs into cancer cells potentially overcoming some important resistance characteristics (in collaboration with Dr. R. Gref, ISMO, CNRS, France).A6

A2. Negatively charged cyclodextrins (NCCDs)  clickable

α, β and γ-CDs modified with iminodiacetic acid groups have been synthesized,A7 which have the ability to coordinate with metal ions. Particularly, these derivatives (Figure A4) coordinate with Gd(III) and fast exchanging water molecules and form metal clusters that display high magnetic relaxivity values, especially at high magnetic fields (100 MHz), thus they exhibit significantly higher relaxivity characteristics compared to MRI contrast agents presently in use.A7-8

Figure A4. The negatively charged, polycarboxylated γ-CD in complex with four Gd(III) ions (PM3 calculations, Dr. Y. Lazarou). A7

A3. Cyclodextrins modified with photoactive groups: Focus on cyclodextrin-based multimodal photosensitizer-drug transport and release molecular systems clickable

Photodynamic Therapy (PDT) is used for selectively treating neoplasmic lesions: photosensitizers (PS, usually porphyrin molecules) are delivered systemically and are preferentially accumulated in the tumor. Illumination by light of the appropriate wavelength generates reactive oxygen species (ROS), which are highly deleterious to the tumor cells. CDs linked with photosensitizer units can be used as molecular transporters that combine the drug inclusion properties in the CD cavity with photo-triggered properties of the attached photoactive unit (multimodal CDs).A9-14 As an immediate consequence, CD-porphyrin covalent conjugation could enhance the aqueous solubility of the porphyrin photosensitizer and promote its de-aggregation as well as increase its fluorescence intensity and lifetime. The ultimate test of these systems would be their ability to become internalized by cells and display a localization preference into a certain subcellular compartment, readily imaged due to the inherent fluorescent reporting property of the porphyrin moiety.

Several such systemsA9-14 have been studied recently in the laboratory include: (i) the molecular encapsulation of a NO photodonor guest in a porphyrin-CD conjugate (mTHPP-CD) that causes efficiently phototoxic effects due to the combined action of NO and ROS in a melanoma cell line (Figure A5a, in collaboration with Prof. S. Sortino, U. Catania, Italy);A9 (ii) an SNO-derivative that can produce NO but also carry a drug in its cavity (Figure A5b);A10 (iii) The porphyrin-CD conjugate (mTHPP-CD, Figure A6a) that carries tamoxifen (NDMTAM, an anticancer agent) is found localized in subcellular endosome compartments of cancer cells (Figure A6b, upper panels). Upon specific illumination the confining membranes are ruptured and the drug is released from its confinement (photodelivery) (Figure A6, lower panels), causing considerable damage to the specific tumor cells at very small concentrations (Photochemical Internalization, PCI, in collaboration with Prof. K. Berg and Dr. T. Theodossiou, Oslo U., Norway).

Figure A5.  (a) A cyclodextrin-porphyrin conjugate has the ability to encapsulate a tailor-made nitric oxide photodonor guest molecule that fits into the βCD cavity. This molecular system aggregates into a nano-assembly of 16 nm (a supramolecular bichromophoric aggregate), which (i) retains porphyrin fluorescence, thus enabling its imaging into living cells and (ii) is able to generate reactive nitric oxide (RNOS) and singlet oxygen (ROS) under illumination by visible light while it can be internalized in melanoma cells and induce a significant level of photomortalityA9 probably due to the combined action of RNOS and ROS.  (b) A poly SNO-substituted βCD can phorelease NO very fast and cause photodamage. The cavity can also carry a drug showing the bimodal nature of the derivative. A10


Figure A6. On-demand, localized, photoinduced drug release: (a) the cyclodextrin-porphyrin (mTHPP-βCD) conjugate carrying FITC-labeled tamoxifen  (NDTAM-FITC). (b) Confocal microscopy images of MCF7 cells treated with mTHPP-βCD+NDTAM-FITC  (right, upper panel); upon irradiation of the cells, the FITC-labeled tamoxifen drug (green) is released (right, lower panel). The confinement of the drug in the cavity of the cyclodextrin-porphyrin conjugate is additionally proven by the observation of FRET effect between the FITC and porphyrin chromophores. A13

A4. Cyclodextrin dimers and cyclodextrins modified with carbohydrate groups (glycoclusters) clickable

Dimers of cyclodextrins were synthesized efficiently using the Staudinger ligation approach in aqueous-organic media. The homo-or heterodimers were found to possess cavities active for molecular transport.A15 On the other hand, attachment of recognition sugars in the primary side of cyclodextrins resulted in formation of glycoclusters able to attach on bacterial membranes, thus allowing for possible biomedical applications.A16-17

  1. Cationic Cyclodextrins: Cell Penetrating Agents and Other Diverse Applications. K. Yannakopoulou, Drug Del. Sci. Tech. 2012, 22, 243-249 (review article)
  2. Synthesis and characterization of per(6-guanidino-6-deoxy)cyclo­dextrins and studies of their effect on DNA. N. Mourtzis, K. Eliadou, C. Aggelidou, V. Sophianopoulou, I. M. Mavridis, K. Yannakopoulou, Biomol. Chem. 2007, 5, 125-131.
  3. Synthesis, characterisation, and remarkable biological properties of cyclodextrins bearing guanidino-alkylamino and aminoalkylamino groups on their primary side. N. Mourtzis, M. Paravatou, M. Mavridis, M. L. Roberts, K. Yannakopoulou Chem. Eur. J. 2008, 14, 4188-4200.
  4. Symmetry requirements for the effective blocking of pore-forming toxins: Comparative study with α-, β-, and γ-cyclodextrin derivatives. K. Yannakopoulou, Jicsinszky, C. Aggelidou, N. Mourtzis, T. M. Robinson, A. Yohannes, E. M. Nestorovich, S. Bezrukov, V. A. Karginov, Antimicrob. Agents Chemother. (AAC), 2011, 55, 3594-3597.
  5. Designed positively charged cyclodextrin hosts with enhanced binding of penicillins as carriers for the delivery of antibiotics: the case of oxacillin. Agnes, A. Thanassoulas, P. Stavropoulos, G. Nounesis, G. Miliotis, V. Miriagou, E. Athanasiou, G. Benkovics, M. Malanga, K. Yannakopoulou, Int. J. Pharm. 2017, 531, 480-491.
  6. Positively charged cyclodextrins as effective molecular transporters of active phosphorylated forms of gemcitabine into cancer cells. Rodriguez-Ruiz, A. Maksimenko, G. Salzano, M. Lampropoulou, Y. G. Lazarou, V. Agostoni, R. Gref, K. Yannakopoulou, Scientific Reports 2017, 7: 8353.
  7. Novel Polycarboxylated EDTA-Type Cyclodextrins as Ligands for Lanthanide Binding: Study of Their Luminescence, Relaxivity Properties of Gd(III) Complexes, and PM3 Theoretical Calculations. D. Maffeo, M. Lampropoulou, M. Fardis, Y. G. Lazarou, I. M. Mavridis, D. A. I. Mavridou, E. Urso, H. Pratsinis, D. Kletsas, K. Yannakopoulou, Biomol. Chem. 2010, 8, 1910-1921.
  8. Anionic cyclodextrins as versatile hosts for pharmaceutical nanotechnology: Synthesis, drug delivery, enantioselectivity, contrast agents for MRI. I. M. Mavridis, K. Yannakopoulou, J. Pharm. 2015, 492, 275-290. (Review article)
  9. A Multifunctional Bichromophoric Nanoaggregate for Fluorescence Imaging and Simultaneous Photogeneration of RNOS and ROS. Fraix, A. R. L. Gonçalves, V. Cardile, A. C. E. Graziano, T. A. Theodossiou, K. Yannakopoulou, S. Sortino Chem. Asian J. 2013, 8, 2634-2641.
  10. S-Nitroso-β-Cyclodextrins as Novel Bimodal Carriers: Preparation, Detailed Characterization, Nitric Oxide Release and Molecular Encapsulation. Piras, T. A. Theodossiou, M. D. Manouilidou, S. Sortino, K. Yannakopoulou Chem. Asian J. 2013, 8, 2768-2778.
  11. Protoporphyrin IX-β-Cyclodextrin Bimodal Conjugate: Nanosized Drug Transporter and Potent Phototoxin. Ch. Aggelidou, T. A. Theodossiou, K. Yannakopoulou Photobiol. 2013, 89, 1011-1019.
  12. Photophysics and ex vivo biodistribution of β-cyclodextrin–meso-tetra(m-hydroxyphenyl)­porphyrin conjugate for biomedical applications. V. Kirejev, A. R. Gonçalves, C. Aggelidou, I. Manet, J. Mårtensson, K. Yannakopoulou, M. B. Ericson Photobiol. Sci. 2014, 13, 1185-1191.
  13. Photochemical Internalization of Tamoxifens Transported by a “Trojan-Horse” Nano¬conjugate into Breast-Cancer Cell Lines. T. A. Theodossiou A. R. Goncalves, K. Yannakopoulou, E. Skarpen, K. Berg Chem. Int. Ed. 2015, 54, 4885-4889.
  14. A versatile δ-aminolevulinic acid (ΑLA)-cyclodextrin bimodal conjugate-prodrug for PDT applications with the help of intracellular chemistry. Ch. Aggelidou, T. A. Theodossiou, A. R. Gonçalves, M. Lampropoulou and K. Yannakopoulou, Beilstein J. Org. Chem. 2014, 10, 2414–2420.
  15. Staudinger ligation towards cyclodextrin dimers in aqueous/organic media. Synthesis, conformations and guest-encapsulation ability. D. Manouilidou, Y. G. Lazarou, I. M. Mavridis, K. Yannakopoulou Beilstein J. Org. Chem. 2014, 10, 774-783.
  16. Synthesis and characterisation of novel glycoclusters based on cell penetrating heptakis(6-aminoethylamino-6-deoxy)-β-cyclodextrin. M. Lampropoulou, K. Yannakopoulou, Incl. Phen. Macrocycl. Chem. 2011, 70, 345-352.
  17. Synthesis of cyclodextrin derivatives with monosacharides and their binding with ampicillin and selected lectins. Lampropoulou, K. Misiakos, M. Paravatou, I. M. Mavridis, K. Yannakopoulou, ARKIVOC, 2015 (iii) 232-243.