Studies On Solid Lipid Nanoparticles Based Gene Vectors

In recent years, the potential prospect of gene therapy as the way to replace the disfunctional gene and to tackle cancer has been widely investigated. The main course of gene therapy is the acquisition and high transfection efficiency of target gene. The success of gene therapy rests on development of a vector that can selectively and efficiently deliver gene to the target cells with minimal toxicity. Gene transfer vectors can be divided into two categories: viral and non-viral agents. Though most of the clinical trials (about 65 per cent) involving gene therapy were by means of the viral vectors. While, they suffer from several drawbacks, including limited carrying capacity, problems with safety, such as mutation, carcinogenesis and the elicitation of an immune response that render transgene expression transient, thus, to a certain extent, widely application of them were limited. In contrast, non-viral delivery systems are less toxic, less immunogenic, much safer and easy to large-scale production, however, the common weakness of them is low transfection efficiency, especially in the presence of serum protein. To overcome these limitations as far as possible, the novel gene vectors which have safe and high transfer ability, are badly required. At present, the gene delivery systems based on nano-carriers have gained increasing attention because of their advantages (such as less toxic, less immunogenic and relatively high transfection efficiency). In this field, solid lipid nanoparticles (SLNs) has been particularly appealing as a new kind of biocompatible and biodegradable gene nano-carrier, because SLNs may offer a number of technological advantages. These include excellent storage stability, a relatively easy production, the possibility of steam sterilization and lyophilization, and large scale production. Moreover, SLN are obtained by using physiologically well-tolerated ingredients already approved for pharmaceutical applications in humans and show low toxicity when injected intravenously.In the present study, pEGFP was taken as reporter gene to be carried by SLNs composed of biocompatible lipids, such as stearic acid (or ATO888) and soya lecithin. The three solid lipid nanoparticles based gene vectors were prepared or assembled by different technologies: 1) cationic SLNs /pDNA binary complex; 2) Anionic protamine-pDNA complex loaded SLNs; 3) Anionic SLNs supported on protamine/DNA complexes. The main methods and results were as follows:1. Selection and preparation of model geneE.Coli (DH 5-α) was employed to amplify the reporter genes and therapeutic genes for our study. Plasmid DNA was isolated and purified from the cells using the Qiagen Giga Endo-free plasmid purification kit. DNA concentration and purity were quantified by UV absorbance at 260 nm and 280 nm. The structural integrity and topology of purified DNA was analyzed by agarose gel electrophoresis.2. Studies on cationic solid lipid nanoparticles/pDNA binary complexCationic liposomes have several advantages including simplicity for use, ease of large-scale preparation, and lack of immunogenicity. However, the stability of cationic liposomes/DNA complexes during storage is limited. In this study, cationic SLNs was designed to overcome the disadvantage of cationic liposomes. Cationic SLNs was prepared by double emulsion method with cationic surfactant, cetyltrimethylammonium bromide (CTAB), which was used to provide positive charge on the surface of nanoparticles, and the report gene was condensed using the cationic SLNs via electrostatic force leading to the formation of the SLNs-pDNA complex. The electric quantity of the system was modified by addition of various concentrations of Ca²⁺. The resulting complex was uniform spherical with an average size of 91.6±5.3nm. Average cell viabilities were between 80 and 110% of control at the concentrations studied. The nuclease degradation test results confirmed that the pDNA could be protected considerably under physiological conditions. DNA release behavior from binary complex in vitro was in accord with Weibull model (lnln[1/(100-R/100)] = -0.4482lnt+ 0.1329, r=0.9926). The gene transfection experiment in vitro suggested that SLNs-pDNA complex could transfer the loaded gene into COS-7 cells. Compared with naked pDNA, it was demonstrated that the SLNs-pDNA complex exhibit a high transfection efficiency to cells. After transfection for 24 h, the results indicated that transfection efficiency of SLNs-pDNA complex was lower than that of lipofectamine/DNA in COS-7 cells, while, higher transfection efficiency was obtained at 48 h.3. Studies on anionic protamine-pDNA complex loaded SLNsThough many of these cationic vectors perform well in vitro in reduced serum conditions, most of them suffer from serious drawbacks or lose their efficiency when tested in vivo In a systemic application, it tends to their aggregation and accumulation in the "first pass organs" such as lungs (consequently causing pulmonary embolism), liver and spleen, and finally opsonization and clearance by the reticuloendothelial system (RES), limiting their therapeutic applications. Therefore, well-defined and negatively charged gene delivery systems are needed for systemic gene delivery. To prepare nonviral vector of solid lipid nanoparticles carrying protamine-pDNA complex (PD-SLN), in which the core of plasmid pDNA condensed by polycation, is encapsulated by lipids. Electrophoretic analysis suggested that PD-SLN could protect the pDNA from nuclease degradation and intensive agitation. PD-SLN maintained sustained-release of pDNA for several days in vitro. The present study is an attempt at investigating the negative charged solid lipid nanoparticles as the non-viral gene vector. While, the particle size and entrapment efficiency were not desirable, a reasonable size distribution was followed by lower entrapment efficiency, 231±13.7nm and 41.5±3.62% respectively; or improved entrapment efficiency would result in larger particle sizes, 627±22.9nm and 86.5±5.28%, respectively. Thus, we are interested in designing anionic ternary nanoparticles via electrostatic attraction instead of encapsulation to remedy the limitations of the PD-SLN.4. Anionic SLNs supported on protamine/DNA complexesThe study result of PD-SLN was not very satisfactory, hence, a new design idea about ternary nanoparticles was proposed. The small SLNs were prepared by a modified film dispersion-ultrasonication method with with an average size of (19.2±5.5) nm, adsorption of anionic SLNs onto the binary complexes (165±28.1nm) was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7±10.6) nm, the fluorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efficiency of (96.75±1.13) %. The results of TEM and CD could be considered as the formation evidences of the ternary nanoparticles. The protection from enzymatic degradation with ternary nanoparticles and binary complexes showed average over 85% and 60% of total DNA recovery, there was a significant difference between the two groups (p < 0.05). The in vitro release studies of DNA from binary complexes and ternary nanoparticles were carried out in phosphate buffer (pH 7.4). The release of DNA from ternary nanoparticles followed the Ritger-Peppas equation and could be modeled by the following equation: ln R = 0.6038 ln t + 1.5292 ( r = 0.9942). Compared with binary complexes and lipofactamine, it was demonstrated that ternary nanoparticles exhibit a low cytotoxicity to A549 cells within the monitored period of 48h (p<0.05). The transfection efficiency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, almost equal to that of lipofectamine/DNA complexes revealed by inversion fluorescence microscope observation.Solid lipid nanoparticles could be prepared easily with small particle sizes, low cytotoxicity and high protection to pDNA. In vitro studies have showed that the complex could be a promising non-viral nano-device, which has the potential to make in vivo gene therapy achievable.