Gene therapy, i.e.
, the expression of genetic material with therapeutic activity in cells, holds great potation for the treatment of human diseases [1
]. A gene delivery system, of either viral or non-viral vector, must be used to carry the foreign gene into the target cell. Despite the high transduction efficiency of viral vectors which are derived from viruses by the use of recombinant DNA techniques, their clinical potential should be fully understood in terms of issues related to production, safety and immune response that need to be addressed [2
]. The safety concerns regarding the use of viral vectors in humans make non-viral delivery systems an attractive alternative. The non-viral vectors have recently gained increasing attention due to their stability, safety, ease of preparation, and are easily manufactured for large-scale production for treatment of numerous acquired or inherited human diseases [3
]. Unfortunately, non-viral gene delivery vehicles often affect cell viability and have poor transfection efficiency.
The application of non-viral gene delivery vectors, including liposomes, cationic polymers and polymeric nanoparticles (NPs), could reduce or avoid immunogenicity and associated risks of toxicity. The versatility for formulation, sustained release properties, sub-cellular size and biocompatibility with tissues and cells make nanoparticles a promising system to achieve ideal gene tranfection [5
]. The nanoparticles prepared by biocompatible and biodegradable poly (d
-glycolide) (PLGA) and poly (d
-lactide) (PLA) polymers have attracted much attention because of their favorable physicochemical characteristics in terms of safety, stability, the relative ease of large-scale production, and lack of intrinsic immunogenicity that make them suitable candidates for gene delivery application [6
]. These biodegradable polymers undergo hydrolysis upon implantation into the body, forming biologically compatible and metabolizable moieties (lactic acid and glycolic acid) that are eventually removed from the body by the citric acid cycle [5
]. However, an important problem with PLA is inadequate interaction between the polymers and cells, and these polymeric NPs can be easily displaced by serum proteins, which can lead to aggregation of NPs. Incorporation of additional excipients such as polyethylene glycol (PEG) has been attempted as a method to prevent the generation of an extremely acidic microenvironment inside the NPs on polymer degradation [9
]. PEG has been used to coat the PLA NPs, and could often improve the solubility of the NPs, minimize their aggregation, reduce their interaction with proteins in the physiological fluid, and finally, produce a shielding effect that counteracts effective DNA complexation and steric stabilization to the gene vectors’ surfaces to improve transfection efficiency [10
]. Because the PLA-PEG block copolymer has an amphiphilic character, it forms polymeric micelles in an aqueous milieu, the core is surrounded by a palisade of tethered PEG chains with an appreciably stretched conformation [12
] that have predominant characteristics such as a long blood circulation time, biodistribution and lower interactions with the reticuloendothelial system (RES) [10
]. Generally, plasmid DNA is encapsulated into PLA particles using the common water-in-oil-water (W/O/W) double emulsion/solvent evaporation method in order to protect plasmid DNA and control the release process [11
]. However, these preparation methods can not guarantee the integrity and can even result in the degradation of DNA under the ultra-sonication or high speed homogenization which is necessary in the encapsulation process to obtain smaller particle sizes [14
]. To protect the DNA cargo, one approach in the application of PLGA NPs for nucleic acid delivery uses adsorption of the anionic DNA molecules onto cationic NPs by the use of cationic surfactants, like cetyltrimethyl ammonium bromide (CTAB), and 3,2′-dimethyl-4-aminobiphenyl (DMAB) in the formulations [15
]. In our previous studies, Zou et al.
also investigated how gene loaded cationic PLA-PEG NPs modified by CTAB could successfully transfect a gene into HeLa cells even in the presence of serum [16
]. However, there is no doubt that introduction of cationic surfactants would generate cytotoxicity. The polar and hydrophobic domains of cationic surfactants may have dramatic effects on both transfection and toxicity levels [17
On the basis of PLA-PEG, as previously, in order to avoid the use of cationic surfactants, here we try to design a biodegradable tri-block copolymer poly(lactic acid)-poly(ethylene glycol)-poly(l
-lysine) (PLA-PEG-PLL), which combines the characters of cationic polymer PLL, biodegradable polymer PLA and PEG, which was selected as the shell-forming segment due to its physicochemical characteristics, including high water solubility and significant chain mobility as well as its low toxicity [12
]. The cationic polymer, poly(l
-lysine) (PLL) has been widely applied in gene delivery vectors. The primary
-amine groups of lysine in PLL could electrostatically interact with negatively charged DNA [18
] and help to improve the affinity to proteins and cells. Based on the electrostatic binding with the tumor cell, PLL-based cationic and biodegradable polymeric micelles are expected to permeate tumor cells more feasibly [19
]. In addition, due to the large number of active functional groups with amino, PLL could be modified with various kinds of ligands to achieve active targeting to tissues and cells.
Compared with various kinds of polymers, cationic polymer PLL was biodegradable but exhibited low transfection efficiency. PEI was considered as one of most efficient currently-available transfection reagents, but this molecule is not biodegradable and relatively cytotoxic. Cationic PLA-PEG NPs modified with cationic surfactants would induce cytotoxicity, while PLA-PEG-PLL NPs were biodegradable, induced low cytotoxicity and possessed a positive charge without any cationic surfactants. Based on these considerations, the main goal of the present work was to synthesize PLA-PEG-PLL and explore its applicability and feasibility as a non-viral vector for gene transport.