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Novel multifunctional cyclodextrin-based nanocarriers for drug encapsulation and delivery as a strategy to overcome current therapeutic drawbacks

Final Report Summary - CYCLON (Novel multifunctional cyclodextrin-based nanocarriers for drug encapsulation and delivery as a strategy to overcome current therapeutic drawbacks.)

www.itn-cyclon.eu
INTRODUCTION.
There is a high demand for the development of new drug delivery strategies to combat major diseases in our society, particularly cancer. Current treatments are based on high efficacy drugs, however their non-selective uptake by both normal and tumor cells as well as the development of multidrug resistance, constitute major hurdles. Passive targeting, involving enhanced permeability and retention effect, is sought using high molecular weight drug carriers. This would allow transfer and accumulation of drugs on tumor sites while active targeting could be materialized with suitable functionalities. Cyclodextrins (CDs), are biocompatible and biodegradable oligosaccharide nanocages, known to improve the solubility, stability and bioavailability of drugs. Scattered literature reports incidents that CDs may constitute potential means to overcome certain forms of multidrug resistance or to effectively deliver photosensitizing anticancer drugs preserving their photodynamic properties. Finally, photodynamic therapy (PDT), aims at selectively killing neoplastic lesions by the combined action of a photosensitizer and controlled use of visible light.

OBJECTIVES
The CYCLON project has aimed to prepare and study diverse families of new CD derivatives to build an arsenal of CD-based drug delivery nanosystems encompassing many cavities in a nm-sized vehicle, high drug loading capacity, improved permeability and retention effect, enhanced targeting, complete biocompatibility and ability to evade resistance mechanisms, in combination with photoactivated functionalities/moities. As an integral part of its objectives CYCLON was committed to the training of nine early stage and four experienced young researchers.

DESCRIPTION OF WORK PERFORMED SINCE THE BEGINNING OF THE PROJECT
RESEARCH: The network is highly specialized in CD chemistry, photochemistry, in vitro drug evaluation and in vivo applications. Specifically, the groups of CYCLON are experts in synthesis of cyclodextrin derivatives (Dr. K. Yannakopoulou, NCSR "Demokritos", Greece; Dr. E. Fenyvesi, CycloLab, Hungary; Prof. A. Vargas Berenguel, University of Almeria, Spain) photochemistry and photodynamics of supramolecular systems (Prof. A. Douhal, Univ. Castilla La Mancha, Spain; Dr. S. Monti, CNR/ISOF Bologna; Prof. S. Sortino, Univ. Catania, Italy), nanoparticles and drug delivery systems [Dr. R. Gref, CNRS Paris, expert on all aspects related to drug delivery; Prof. S. Sortino, Univ. Catania, with emphasis in delivery of Reactive Oxygen and Nitric Oxide Species (ROS and RNOs)] and pharmacology/applications (Prof. T. Loftsson, U. of Iceland; Prof. M. Ericson, U. Gothenbourgh, Sweden, with focus on skin delivery and PDT). The groups have synthesized diverse families of new CD derivatives, which alone or in combination with nano-Metal Organic Frameworks (nanoMOFs) provide an arsenal of drug delivery nanosystems with a variety of architectures, emphasizing on multifunctionality, enhanced encapsulation, improved delivery, biocompatibility and targeting. Selected examples include: a nanoplatform with four-in-one photoresponsive functionalities based on biocompatible polymeric nanoparticles efficiently delivered different photosensitizers in cells and induced amplified cell mortality (Figure 1a, [1]). Nanosized biodegradable and biocompatible nanoMOFs were found to be ideal carriers for the antiviral AZT-mono- and –triphosphate, with entrapment efficiencies close to 100% that penetrate inside major HIV target cells and release their cargo of the active AZT-TP, efficiently protecting against HIV infection (Figure 1b, [2]). NanoMOFs decorated with modified CDs displayed enhanced stability and drug loadability. The method has been patented by CNRS and CycloLab (Figure 1c). Photoresponsive polymer nanoparticles have encapsulated fluorophore/photochrome dyads and nitric oxide photodonors and transported their cargo across cell membranes. Two guests could be used simultaneously to activate fluorescence and release nitric oxide under optical control (Figure 1d, [3]). A supramolecular nanoaggregate composed of a synthesized porphyrin-beta-CD conjugate and a tailored NO photodonor guest was effectively internalized and imaged inside cancer cells while it induced a good level of cellular death under the exclusive control of visible light. (Figure 1e, [4]). Two citric acid crosslinked gamma-CD oligomers prepared by green methods can bind with monomerized anticancer drug doxorubicin (DOX) 1–2 orders of magnitude stronger than gamma-CD alone. Imaging showed a similar drug distribution within MCF-7 cells without any DOX degradation (Figure 1f, [5]). An engineered hydrogel has been developed by spontaneous self-assembly of a beta-CD polymer, a hydrophobically modified dextran and a tailored nitric oxide (NO) donor. The effective, light-dependent bactericidal activity of this NO photo-releasing platform against E. coli colony was demonstrated (Figure 1g, [6]). Mono- and multi-SNO-βCD released NO thermally or photochemically in a controlled fashion, and were able to carry a chemotherapeutic drug (tamoxifen) (Figure 1h, [7]). More results with multimodal nanoplatforms with intriguing properties have been published [8] or are in preparation. Many prototype systems have been studied regarding their photochemistry and photodynamic properties, stability, stoichiometry, lifetime of excited species in confined space and other spectroscopic properties (for example Figure 1i, [9-12] and many more), providing the background for understanding the systems. CD complexes seem to form aggregates that enhance the aqueous solubility of drugs and regulate their release, whereas fluorescent CDs and conjugates have demonstrated increased skin penetration capacity, as shown by pharmaceutical technology and two photon microscopy and other methods (for example [13,14]). Fluorescent CDs developed by CycloLab (http://www.cyclolab.hu/product1_5.html) now a new line of commercial products, have been applied for cell permeation studies and delivery experiments in vitro (Figure 1j). Out of fifty journal publications, fourteen are joint articles, produced by collaborations that were initiated during secondments of the MC fellows. More articles are currently in the submission stage or in preparation.
TRAINING: Four workshops, four Summers Schools and one International conference (NanoPDT), were organized (www.itn-cyclon.gr) in the frame of CYCLON. The research training program of ESRs and ERs in a highly collaborating, multidisciplinary and application oriented approach has resulted already in four PhD degrees awarded and five more scheduled. Four ERs have been employed in research positions and one ESR in a private company.

Figure 1a 1b 1c 1d 1e 1f 1g 1h 1i 1j

IMPACT
The CYCLON project has developed a collection of new multifunctional and versatile drug delivery platforms based on specifically modified cyclodextrins and nanoMOFs or their combination. In vitro and ex vivo tests gave very promising results on the effectiveness of several systems. The research constitutes a significant contribution in the field of nanomedicine, addressing simultaneously many drug delivery issues and proof of principle (delivery and effectiveness) has been demonstrated in several cases. Further, nine young researchers and four post-docs were trained so as to embark on academic or industrial research careers. Four PhD degrees have been already awarded and five more expected to be awarded in 2014. Five persons have found employment.

SELECTED REFERENCES
[1] An engineered nanoplatform for bimodal anticancer phototherapy with dual-color fluorescence detection of sensitizers, A., N. Kandoth, I. Manet, V. Cardile, Adriana C. E. Graziano, R. Gref and S. Sortino, Chem. Commun., 2013, 49, 4459.
[2] a) Impact of phosphorylation on the encapsulation of nucleoside analogues within porous iron(III) metal–organic framework MIL-100(Fe) nanoparticles, V. Agostoni, R. Anand, S. Monti, S. Hall, G. Maurin, P. Horcajada, C. Serre, K. Bouchemal, R. Gref, J. Mater. Chem. B, 2013, 1, 4231. b) Towards an improved anti-HIV activity of NRTI via Metal Organic Framework nanoparticles, V. Agostoni, T. Chalati, P. Horcajada, H. Willaime, H. Chacun, T. Baati, S. Hall, G. Maurin, H. Chacun, K. Bouchemal, C. Martineau, F. Taulelle, F. P. Couvreur, C. Roger-Kreuz, P. Clayette, R. Anand, S. Monti, C. Serre, R, Gref., Adv. Healthcare Mater.2013 DOI:10.1002/adhm.201200454
[3] Photoinduced fluorescence activation and nitric oxide release with biocompatible polymer nanoparticles, E. Deniz, N. Kandoth, A. Fraix, V.Cardile A. C. E. Graziano, D. Lo Furno, R. Gref, F. M. Raymo, S. Sortino, Chem. Eur. J., 2012, 18(49), 15782.
[4] A multifunctional bichromophoric nanoaggregate for fluorescence imaging and simultaneous photogeneration of RNOS and ROS, A. Fraix, A.R. L. Gonçalves, V. Cardile, A. C. E. Graziano, T. A. Theodossiou, K. Yannakopoulou, S. Sortino, Chem. Asian J. 2013, 8(11), 2634.
[5] Citric acid -γ-cyclodextrin crosslinked oligomers as carriers for doxorubicin delivery, S. Monti, R. Anand, M. Malanga, I. Manet, F. Manoli, K. Tuza, A. Aykaç, C. Ladaviére, E. Fenyvesi, A., Vargas-Berenguel, R. Gref, Photochem. Photobiol. Sci., 2013, 12, 1841.
[6] A NO photoreleasing supramolecular hydrogel with bactericidal action, N. Kandoth, J. Mosinger, R. Gref, S. Sortino, J. Mater. Chem. B, 2013, 1(28), 3458.
[7] S-Nitroso-beta-cyclodextrins as new bimodal carriers: preparation, detailed characterization, nitric-oxide release, and molecular encapsulation, L. Piras, T. A. Theodossiou, M. D. Manouilidou, Y. G. Lazarou, S. Sortino, K. Yannakopoulou, Chem. Asian J., 2013, 8(11), 2768.
[8] A host-guest supramolecular complex with photoregulated delivery of nitric oxide and fluorescence imaging capacity in cancer cells, N. Kandoth, M. Malanga, A. Fraix, L. Jicsinszky, É. Fenyvesi, T. Parisi, I. Colao, M.T. Sciortino and S. Sortino, Chem. Asian J., 2012, 7 (12), 2888.
[9] Femtosecond studies of a water soluble porphyrin derivative in chemical and biological nanocavities, Y. Wang, B. Cohen, L. Jicsinszky, and A. A. Douhal, Langmuir, 2012, 28(9), 4363.
[10] Photocontrolled binding of artemisinin to a bis(β-cyclodextrin) bearing azobenzene on the primary face, R. Anand, F. Manoli, A. Vargas-Berenguel, S. Monti, J. Drug Del. Sci. Tech., 2012, 22(3), 266.
[11] Femto- to micro-second photobehavior of photosensitizer drug trapped within a cyclodextrin dimer, Y. Wang, B. Cohen, A. Aykaç, A. Vargas-Berenguel, A. Douhal, Photochem. Photobiol. Sci., 2013, DOI: 10.1039/C3PP50179E.
[12] beta-Cyclodextrin polymer nanoparticles as carriers for Doxorubicin and Artemisinin: a spectroscopic and photophysical study, R. Anand, F. Manoli, I. Manet, S. Daoud-Mahammed, V. Agostoni, R. Gref and S. Monti, Photochem. Photobiol. Sci., 2012, 11 (8), 1285.
[13] A permeation method for detection of self-assembly of doxorubicin in aqueous environment, Z. Fülöp, R. Gref, T. Loftsson, Int. J. Pharm., 2013, 454(1), 559.
[14] a) Multiphoton microscopy- a powerful tool in skin research and topical drug delivery science, V. Kirejev, S. Guldbrand, J. Borglin, C. Simonsson, M. B. Ericson, J. Drug Del. Sci. Tech. 2012, 22(3), 250; b) V. Kirejev, N. Kandoth, R. Gref, M. B. Ericson and S. Sortino, A Polymer-Based Nanodevice for the Photoregulated Release of NO with Two-Photon Fluorescence Reporting in Skin Carcinoma Cells, J. Mater. Chem. B. 2013, accepted
CONTACT DETAILS : Dr. K. Yannakopoulou, Coordinator (dyanna@chem.demokritos.gr) NCSR “Demokritos, Aghia Paraskevi 15310, Athens, Greece.