International Journal of Pharmaceutics 446 (2013) 176–182 Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics jo ur n al homep age: www.elsevier.com/locate/ijpharm Pharmaceutical nanotechnology Incorporation of carbon nanotubes into a gelatin–catechin conjugate: Innovative approach for the preparation of anticancer materials Giuseppe Cirillo a,b,∗ , Orazio Vittorio c , Silke Hampel b , Umile Gianfranco Spizzirri a , Nevio Picci a , Francesca Iemma a a Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, I-87036 Arcavacata di Rende, CS, Italy b Leibniz Institute for Solid State and Materials Research Dresden, PF 270116, D-01171 Dresden, Germany c NEST Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro, I-56126 Pisa, Italy a r t i c l e i n f o Article history: Received 20 November 2012 Received in revised form 30 January 2013 Accepted 8 February 2013 Available online 19 February 2013 Keywords: Anticancer activity Carbon nanotubes Catechin Gelatin Polymer composites a b s t r a c t A new hybrid material made of gelatin, catechin and carbon nanotubes was prepared by the non-covalent incorporation of carbon nanotubes (CNTs) into a gelatin–catechin covalent conjugate. The composite materials was tested by means of determination of the dispersion stability in water and the functional- ization degree was assessed by the Folin–Ciocalteu method, finding a 0.9 mg of CT/g of protein conjugate. Subsequently, the complete retention of the antioxidant properties of the flavonoid after incorpora- tion into the composite was proved by DPPH and ABTS assays and IC 50 values of 5.74 mg mL -1 and 0.39 mg mL -1 were recorded. The presence of CNT into the materials did not interfere with the scav- enging activities of the catechin. Finally, the anticancer activity on HeLa cancer cells was evaluated and a considerable increase in the therapeutic activity of the flavonoid was recorded moving from the free to the conjugated form in the presence of CNT, while in absence of CNT a reduction of the efficiency was observed. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Gelatin is a water-soluble functional protein of high interest and value obtained by partial hydrolysis of collagen, the main fibrous protein constituent in animal skins, bones, tendon, and loose connective tissues (Djagny et al., 2001; Gómez-Guillén et al., 2011). Mainly because of its gel-forming and viscoelastic proper- ties, gelatin has been used in food science as clarification agent, stabilizer, and protective coating material, in pharmaceutical and medical field for the manufacture of pharmaceutical capsules, oint- ments, cosmetics, tablet coating, and emulsions (Gómez-Guillén et al., 2011; Young et al., 2005). Furthermore, researches in bioma- terials science have explored new materials to be used in medical devices, and in this context, gelatin has been proposed as attrac- tive candidates for tissue engineering (Palsson and Bhatia, 2004). Examples of these applications are the use as plasma substitute and a positive benefit of gelatin on bone turnover makers and joint health (Booth and Highley, 2010; Jegal et al., 2011). ∗ Corresponding author at: Department of Pharmacy, Health and Nutrition Sci- ences, University of Calabria, I-87036 Arcavacata di Rende, CS, Italy. Tel.: +39 0984 493011; fax: +39 0984 493011. E-mail addresses: giuseppe.cirillo@unical.it, giu.cirillo@gmail.com (G. Cirillo). Despite of the reported wide applicability of gelatin, the bio- logical activity of gelatin was assessed as “zero” by the research of Bender and Miller, mainly because of the absence of the “essen- tial” tryptophan (Bender et al., 1953). To overcome this limitation and thus increase the applicability of protein, and gelatin in par- ticular, several different studies have been performed to modify the conformation, structure, and consequently to produce food- grade and pharmaceutical ingredients with improved functional properties through physical, chemical, and/or biological treatments (Fernandes et al., 2011; Pe˜ na et al., 2010; Lai, 2010). In any case, the modification of gelatin are mainly related to the physic chem- ical properties, while only few examples report on the chemical modification with biologically active molecule to impart specific biological properties to the protein (Law and Tung, 2009). One of the most interesting research fields covering the last decade is that of polymer therapeutics, and several different materials have been planned and developed, mainly with the aim to provide tether- ing sites for therapeutic molecules or targeting agents (Greco and Vicent, 2009; Luo et al., 2000). Regarding gelatin, in recent publica- tions, the possibility to obtain a covalent coupling with antioxidant molecules has been proposed and functional protein with enhanced biological activity, e.g. antioxidant, hypoglycemic, and anticancer (Spizzirri et al., 2009; Cirillo et al., 2010). Among these properties, the most interesting is surely the ability to impart anticancer behav- ior to the protein, and in particular, a gallic acid–gelatin conjugate 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.02.023