Cell penetrating peptide tethered bi-ligand liposomes for delivery to brain in vivo: Biodistribution and transfection Gitanjali Sharma, Amit Modgil, Buddhadev Layek, Kanwardeep Arora, Chengwen Sun, Benedict Law, Jagdish Singh ⁎ Department of Pharmaceutical Sciences, College of Pharmacy, Nursing, and Allied Sciences, North Dakota State University, Dept# 2665, Sudro Hall, Room 102A, PO Box 6050, Fargo, ND 58108–6050, USA abstract article info Article history: Received 2 October 2012 Accepted 14 January 2013 Available online 23 January 2013 Keywords: Bi-ligand Liposomes Blood brain barrier Gene delivery Cell penetrating peptide Biocompatibility Targeted nano-particulate systems hold extraordinary potential for delivery of therapeutics across blood brain barrier (BBB). In this work, we investigated the potential of novel bi-ligand (transferrin-poly-L-arginine) liposo- mal vector for delivery of desired gene to brain, in vivo. The in vivo evaluation of the delivery vectors is essential for clinical translation. We followed an innovative approach of combining transferrin receptor targeting with enhanced cell penetration to design liposomal vectors for improving the transport of molecules into brain. The biodistribution profile of 1, 1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine iodide(DiR)-labeled lipo- somes was evaluated in adult rats after single intravenous injection at dose of 15.2 μmoles of phospholipids/kg body weight. We demonstrated that bi-ligand liposomes accumulated in rat brain at significantly (p b 0.05) higher concentrations as compared to the single-ligand (transferrin) or plain liposomes. In addition, the bi-ligand liposomes resulted in increased expression of β-galactosidase(β-gal) plasmid in rat brain tissue in comparison to the single-ligand liposomes. Histological examination of the transfected tissues did not show any signs of tissue necrosis or inflammation. Hemolysis assay further authenticated the biocompatibility of bi-ligand liposomes in blood up to 600 nmoles of phospholipids/1.4×10 7 erythrocytes. The findings of this study provide important and detailed information regarding the distribution of bi-ligand liposomes in vivo and accentuate their ability to demonstrate improved brain penetration and transfection potential over single-ligand liposomes. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Gene therapy is an effective tool for providing lasting and notable treatment for genetic disorders like cystic fibrosis, combined immunode- ficiency syndrome and many cancers that result from the presence of defective genes [1–3]. Considering the remarkable progress in the devel- opment of efficient gene transfer vectors leading to safer transduction and robust transgene expression coupled with increased understanding of the molecular mechanisms of neurological and other CNS disorders, it is rational to believe that gene therapy can play an inevitable role in the treatment of CNS diseases by introducing genes into brain [4,5]. Gene therapy can be used for the treatment of a wide range of CNS disorders like neurodegenerative diseases by increasing the expression of growth factors, anti-apoptotic molecules or antioxidants [5–7]; it can reduce the proliferation of tumor cells by producing anti-angiogenic factors or by down regulating the expression of certain genes using antisense or siRNA [8–11]. However, the major challenge faced by the scientific community lies in the safe and efficient delivery of therapeutic genes to the brain. The formidable challenge of delivering an effective therapeutic agent to brain requires collaborative multidisciplinary efforts that take into consideration the blood brain barrier (BBB) biology as well as the study of basic transport mechanisms for delivery to brain. There are about 100 billion capillaries in the human brain with a sur- face area of approximately 20 m 2 that constitute the BBB [12,13]. The passage of molecules administered via parenteral route to brain is tightly controlled by the brain capillary endothelium [14]. Brain en- dothelial cells have specific receptors for the transport of ligands like transferrin, enzymes, growth hormones, etc. Transferrin receptor mediated transcytosis is the most well characterized system for trans- port of iron to brain [15]. In addition, the transferrin receptors on the surface of brain endothelial cells have been widely explored for targeting various drug delivery systems to brain [16–18].These recep- tors bind to targeting ligands which are then transcytosed across the capillary endothelium and the receptor molecule is recycled [19]. How- ever, the presence of receptor-targeting moiety alone on sterically sta- bilized vector limits the delivery of the vector due to receptor saturation [18,20]. In the light of these details; we followed a dual mechanistic approach for targeting transferrin receptors on brain Journal of Controlled Release 167 (2013) 1–10 ⁎ Corresponding author at: Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA. Tel.: +1 701 231 7943; fax: +1 701 231 8333. E-mail address: jagdish.singh@ndsu.edu (J. Singh). 0168-3659/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2013.01.016 Contents lists available at SciVerse ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel GENE DELIVERY