Synthesis of TAT peptide-tagged PEGylated chitosan nanoparticles for siRNA delivery targeting neurodegenerative diseases Meenakshi Malhotra, Catherine Tomaro-Duchesneau, Satya Prakash * Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Artificial Cells and Organs Research Center, Faculty of Medicine, McGill University, 3775 University Street, Montreal, Quebec H3A 2B4, Canada article info Article history: Received 25 August 2012 Accepted 4 October 2012 Available online xxx Keywords: Chitosan PEG TAT siRNA Nanoparticles Gene delivery abstract Delivery of therapeutic molecules to the brain for the treatment of Neurodegenerative diseases (ND) is a challenging task. This manuscript introduces a novel scheme of synthesizing peptide-tagged poly- ethylene glycol (PEG)ylated chitosan polymer to develop nanoparticles for siRNA delivery for use in ND. Specifically, this manuscript proposes a facile chemoselective conjugation of monomethoxy PEG, at the C2 hydroxyl group of chitosan polymer, with conjugation of PEG to a cell-penetrating peptide, Trans- Activator of Transcription. The synthesized Chitosan-PEG-TAT polymer was used to form the nano- particles of approximately 5 nm, complexing siRNA to be delivered in neuronal cells (Neuro 2a), with no/ minimal toxicity. The various intermediates and the final product formed during the synthesis were characterized using 1 H Nuclear Magnetic Resonance and Fourier Transform Infrared Spectroscopy spectra. The morphological details of the nanoparticles were studied using Transmission Electron Microscopy. The nanoparticles were tested to deliver a functional siRNA against the Ataxin-1 gene in an in-vitro established model of a ND Spinocerebellar ataxia (SCA1) over-expressing ataxin protein. The results indicate successful suppression of the SCA1 protein following 48 h of transfection. Result of this study has potential in ND like SCA, Parkinson’s, Alzheimer’s and others. Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. 1. Introduction Neurodegeneration is characterized by the progressive loss of structure and function of neurons. One among many neurodegen- erative diseases is Spino Cerebellar Ataxia (SCA), characterized by a loss of cells of the cerebellum and spinal cord affecting motor coordination, posture and balance. The international prevalence of SCA is estimated to be 0.3e2.0 per 100,000 [1]. SCA is caused by polyglutamine trinucleotide repeat expansion, CAG repeat and forms an abnormal/mutant protein when the CAG repeat unit is above the threshold level, i.e. approximately 35 repeat units [2]. The abnormal protein then accumulates in the Purkinje cells of the brain and also cytoplasm, dendrites and axonal processes [2].A recent review by our group highlights the different subtypes of SCA and the current therapeutic methods and drugs used to alleviate the symptoms of this disease [2]. The reason for the absence of available medication could be attributed to the genetic problem associated with it. In the case of SCA, the cell production of poly glutamine cannot be halted as it would interfere with the function of the brain cells and, thus, would produce side-effects which may in turn require further treatment. However, certain drugs like Deferipone and Idebenone [3] have reached phase II and III clinical trials, respectively, but are limited to Friedreich’s ataxia (Autosomal Recessive Cerebellar Ataxia). Several other reports on the active and completed clinical trials of other related neurodegenerative diseases can also be found at clinicaltrials.gov. RNA interference technology has been demonstrated as an effective therapeutic modality in vivo for the reduction of patho- logical molecules in neurons, for the treatment of Spinocerebellar Ataxia [4]. The other therapeutic targets that have been successfully inhibited with siRNA include ion channels [5], growth factors [6] growth factor receptors [7] and transcription factors [8]. The currently used drug/therapeutic delivery strategies such as implan- tation of catheters, intra-carotid infusions, surgeries and chemo- therapies are invasive in nature and pose a greater risk of post- surgical complications like fluid retention in the ventricles etc, which lead to fatal side-effects. It has been estimated that up to 98% of the newly developed small molecules do not cross the bloode brain barrier [9]. Moreover, it is challenging to achieve sufficient distribution and diffusion of the therapeutic drug within the brain. Although viral gene therapy vectors are the most efficient vectors * Corresponding author. 3775 University Street, Room 311, Department of Biomedical Engineering, Lyman Duff Medical Building, Montreal, Quebec H3A 2B4, Canada. Tel.: þ1 514 398 2736; fax: þ1 514 398 7461. E-mail address: satya.prakash@mcgill.ca (S. Prakash). Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.10.013 Biomaterials xxx (2012) 1e11 Please cite this article in press as: Malhotra M, et al., Synthesis of TAT peptide-tagged PEGylated chitosan nanoparticles for siRNA delivery targeting neurodegenerative diseases, Biomaterials (2012), http://dx.doi.org/10.1016/j.biomaterials.2012.10.013