This journal is c the Owner Societies 2013 Phys. Chem. Chem. Phys. Cite this: DOI: 10.1039/c3cp51215k Chitosan conjugates for DNA delivery Diana Paiva, a Galya Ivanova, b Maria do Carmo Pereira a and Sandra Rocha* c Graft copolymers of chitosan and maltodextrin were synthesized by reductive amination of a low molecular weight chitosan. The degree of substitution is 70% or above, as quantified by nuclear magnetic resonance spectroscopy, at molar ratios of chitosan glucosamine units and maltodextrin of 1 : 1 and 1 : 5. The high substitution degree generates a water-soluble compound at pH 7.4 by reducing the amino groups of chitosan. In addition, the copolymer self-assembles into spherical nanoparticles with diameters of about 300 nm. The mass polymer/DNA ratios at the isoelectric point are within the range of 3–3.5 for chitosan–maltodextrin nanoparticles as determined by electrophoretic mobility mea- surements. These results confirm that DNA interacts efficiently with the copolymer nanoparticles indicat- ing a potential application of the system for DNA delivery. Introduction Nanoparticles of chitosan and its derivatives are studied in the pharmaceutical field as drug carriers and DNA delivery systems. 1–3 Chitosan is a linear polymer of b-(1-4)-linked- D-glucosamine mono- mers with randomly distributed N-acetyl- D-glucosamine units obtained by partial deacetylation of chitin, a natural polysaccharide found principally in crustacean shells such as shrimps and crabs. 4 It is a non-toxic and natural biodegradable cationic polymer with low immunogenicity and high biocompatibility. 5,6 Other advan- tages of this macromolecule include stability, versatility of physico- chemical properties and the possibility of modification by attaching cell compatible ligands. 5,7 However, problems related to chitosan delivery systems have been reported: the pH dependence (chitosan is insoluble at neutral pH); the control of particle size, shape and polydispersity due to the variability of chitosan molec- ular weight; the limitation of chitosan application in humans due to hypocholesterolemia caused by high doses of chitosan; the in vitro transfection rate of DNA–chitosan nanoparticles is very much dependent on the cell type. 7–9 The systems are typically prepared either by compacting DNA with the cationic polymer or by encapsulating the active principle in the chitosan matrix. Chitosan colloidal systems normally have a size ranging from a few nano- metres to 500 nm. 10–13 They have been used in nonconventional drug delivery for nasal, ocular and peroral administration in order to prolong the contact time and to enhance the drug absorption. 5 There are many factors influencing the gene expression using chitosan systems. 8,14 Kiang et al. tested chitosan molecules with different molecular weights and degrees of deacetylation and determined that these properties play a key role in the optimization of the formulations for DNA transfection. 15 An alternative approach to improve chitosan properties is to chemically modify its structure by grafting other molecules. 16–19 Mathew et al. conjugated folic acid with carboxymethyl chitosan to produce nanoparticles capable of targeting and controlling the release of 5-fluorouracil, an anticancer drug used in chemotherapy. 20 Park et al. suggested grafting dextran to galactosylated chitosan for targeting genes to hepatocytes. 21 Unmodified chitosan was also grafted to dextran molecules of different molecular weights, resulting in compounds with different degrees of substitution and water solubility at different pH values. 22 In this work, chitosan was conjugated to maltodextrin, a non-ionic excipient of D-glucose units linked by a(1-4) glycosidic bonds. Maltodextrins are produced by partial hydrolysis of starch and are classified in terms of DE (dextrose equivalent), having between 3 and 20 DE. They enhance gene expression and have low toxicity. 23 The conjugation of chitosan to maltodextrin (DE 18–20) was achieved by reductive amination and the resultant copolymer was characterized using FTIR and NMR. Nanoparticles of chitosan- graft-maltodextrin copolymers were analysed using dynamic light scattering and electron microscopy techniques. Experimental section Materials The following materials were used in the experiments. Chitosan (Ch) 20 kDa (degree of deacetylation > 92%) was supplied by Altakitin (Portugal). DNA (deoxyribonucleic acid sodium salt a LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr Roberto Frias, 4200-465 Porto, Portugal b REQUIMTE – Departamento de Quı ´mica, Faculdade de Cie ˆncias, Universidade do Porto, 4169-007 Porto, Portugal c Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg SE-41296, Sweden. E-mail: sandra.rocha@chalmers.se; Tel: +46 31 772 2815 Received 20th March 2013, Accepted 16th May 2013 DOI: 10.1039/c3cp51215k www.rsc.org/pccp PCCP PAPER Published on 13 June 2013. Downloaded by Universidade do Porto (UP) on 17/06/2013 10:45:17. View Article Online View Journal