**1. Introduction**

Delivery of a functional DNA to mammalian cells is an attractive approach for genetic manip‐ ulation of the cells in biomedical research as well as in gene therapy for treating critical human diseases. Following delivery to the cytoplasm, a foreign gene enters the nucleus and is tran‐ scribed to the corresponding mRNA, which is subsequently transported to the cytoplasm for translation into a specific protein. However, a gene-silencing element, such as an antisense onligonucleotide or a small interfering RNA blocks the transcription of a target mRNA. Thus, nucleic acid delivery has been an essential tool either to turn on or off the expression of a particular gene in basic research laboratories.

Intensive research in the last three decades led to the development of a number of viral and non-viral vectors. However, an ideal vector in terms of safety and efficacy is still lacking. Synthetic non-viral vectors, such as cationic polymers, lipids and peptides, are relatively safe, but extremely inefficient. On the other hand, viral systems are by far the most effective means of DNA delivery to mammalian cells, but some major limitations including toxicity, immuno‐ genicity, restricted targeting of specific cell types, restricted DNA carrying capacity, produc‐ tion and packaging problems, recombination and high cost, limit their successful applications in basic research and clinical medicine. The effectiveness of a viral particle is the result of its highly evolved and specialized structure basically composed of a protein coat surrounding a nucleic acid core. Such a highly organized structure can prevent viral particles from unwant‐ ed interactions with serum components, while promoting subsequent internalization by cells, escape from endosomes, and release of genetic material from the particle either before or after entering the nucleus. Development of a non-viral approach having the beneficial virus-like

© 2013 Chowdhury; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

properties and lacking the disadvantageous aspects would emerge as the most attractive one for implementation in research laboratories and gene therapy.

A major barrier to the non-viral delivery is low uptake of DNA across the plasma mem‐ brane of a cell owing to the inappropriate and ineffective interactions of the DNA delivery vehicle with the cell membrane. Negatively charged DNA molecules are usually condensed with cationic reagents to allow formation of the complexes carrying net positive charges. The resulting complexes can interact electrostatically with anionic heparan sulfate proteoglycans (syndecans) on cell surface and reach the cytoplasmic side in the form of endosomes through endocytosis [1]. The extremely low pH and enzymes within the late endosomes usually bring about degradation of entrapped DNA and associated complexes. Finally, DNA that survives both endocytic processing and cytoplasmic nucleases must dissociate from the condensed complexes either before or after nuclear translocation through nuclear pore or during cell division.

Many therapeutic applications demand a vehicle with capability of delivering transgene(s) to a selective cell type in order to increase the expression efficacy and alleviate any side effect. A common strategy in non-viral case involves attachment of a targeting moiety onto a poly‐ cation (lipid or polymer) backbone which finally condenses the DNA through ionic interac‐ tions. Targeting moiety can enable the resulting DNA carrier to bind to a receptor, lectin, antigen or cell-adhesion molecule on plasma membrane prior to internalization via endocy‐ tosis or phagocytosis. Polylysine, the first backbone used for gene delivery has been conjugat‐ ed to a diverse set of cell-specific ligands, such as asialoorosomucoid [2], transferrin [3], epidermal growth factor (EGF) [4], mannose [5], fibroblast growth factor (FGF) [6] and anti‐ bodies [7] for targeting, respectively, hepatocytes via asialoglycoprotein receptors, transfer‐ rin receptor-positive cells, EGF receptor-carrying cells, macrophages through membrane lectins, FGF receptor-bearing cells and lymphocytes via surface-bound antigens. In the similar fash‐ ions, polymers like polyethylenimine and liposomes have been coupled to other cell surface receptor-specific ligands in addition to those described above, such as integrin-binding pep‐ tide conjugated onto PEI to target integrins on cell surfaces [8] and vitamin folate conjugat‐ ed onto liposomes through a polyethylene spacer to target folate receptor-bearing cells [9].

Cell adhesion molecules (integrin, syndecan, cadherin, selectin) which are a diverse group of cell surface proteins mediating interactions between cells, and between cells and the ex‐ tracellular matrix, are valuable targets for precise gene delivery to haematopoietic cells, air‐ way epithelial cells, tumor cells and vascular endothelial cells using synthetically designed non-viral vectors [10].

Recently, we have reported on the development of a safe, efficient nano-carrier system of carbonate apatite which can assist both intracellular delivery and release of DNA leading to very high level of trans-gene expression in cancer and primary cells [11-13]. We have also revealed a new approach of organic-inorganic hybrid carrier devised by complexing fibronec‐ tin and E-cadherin-Fc chimera electrostatically with nano-particles of carbonate apatite [14, 15]. Specific recognition to cell surface integrin and E-cadherin molecules through double ligand-coated nano-particles, resulted in synergistic acceleration of transgene delivery and consequential expression into embryonic stem cells. Instead of simultaneous mixing of DNA and cell-adhesive molecules in particle-preparation medium and subsequent incubation, stepwise addition and incubation of DNA and the protein molecules, results in improved DNA loading and decreased particle diameter with ability of recognizing stem cell surface for more efficient transgene delivery. Activation of PKC which might up-regulate both integrin and Ecadherin, enhances transgene expression in mouse embryonic carcinoma cells.
