br Material and methods br Results br

Material and methods
Results
Discussion Clinical gene therapy trials with viral vectors have been in use for treatment of various genetic disorders and diseases, however efficacious, targetable and highly reliable non-viral delivery systems are needed for safe and long-term applications. Despite their low transfection efficiency compared to the viral delivery systems, cationic SLNs may present advantages in immunogenicity, production, purification, cost and stability [42,43]. In this study, we produced cationic SLNs by the microemulsion dilution technique, which is a commonly used approach in our laboratory and in our previous works as well [44,45]. Microemulsions are thermodynamically stable and their formation does not require an input of energy. Depending on the ratios of ingredients, various types of microemulsions form spontaneously [46,47]. Here, we prepared oil-in-water microemulsion system by using Compritol ATO 888 as internal oil phase which is a solid lipid. Pseudo ternary phase diagram was drawn by water titration with components of microemulsion over lipid melting temperature. As seen in Fig. 2, the translucent and thermodynamically stable microemulsion region is indicated on the pseudo ternary phase diagram. This area could be enlarged depending on the structure of the surfactant and the presence or absence of co-surfactants of an oil-in-water system. We tend to use the lowest ratio of S-CoS to avoid possible toxicity effects. A central point in the o/w microemulsion region was selected on the ternary phase diagram due to its clear appearance and stability. Then, selected cationic lipid was incorporated into the oil phase. We used DDAB as a cationic lipid because of its low cost and low toxicity depending on its two-tailed hydrocarbon chains [48,49]. Cationic lipids are needed to obtain positive superficial charged SLNs and further to interact electrostatically with negative charged DNA to form SLN:DNA complexes. Cationic SLNs were prepared as described before to form SLN: p5α-Red complexes. As can be seen in Fig. 3, optimal complex formation ratio was determined by gel retardation assay. The binding efficiency assay showed that SLN:p5α-Red ratio 300:1 (w/w) or higher are necessary to bind pDNA completely to SLN and block the pDNA migration on the agarose gel [50]. Before physicochemical characterization of SLNs and SLN:p5α-Red vectors with optimal complex formation ratio, DNase I protection analysis was determined as well. SLN's capacity to protect DNA from Caspase-6, human recombinant protein of serum nucleases was examined by incubating SLN:p5α-Red vectors (100:1–500:1, w/w) with DNase I enzyme (0.4 IU DNase I/1 μg pDNA) at 37 °C for 30 min, then p5α-Red was decomplexed in presence of SDS 1% and further subjected by gel retardation assay. The condition of released p5α-Red was examined by measuring band densities and the integrity of the vector was compared with a control of untreated p5α-Red. As seen in Fig. 4, naked p5α-Red plasmid was digested totally when it was compared with a control of untreated p5α-Red in lane 2. Lanes 4–8 are respectively SLN:p5α-Red vectors (100:1–500:1, w/w) that were treated with DNase I. We observed that SLN:p5α-Red vector with the ratio of 300:1 (w/w) in lane 6 were able to protect pDNA and increased protection level was not observed at higher ratios. SLN: p5α-Red ratio of 300: 1 (w/w) was determined as the optimum ratio when considering the need for low SLN content in terms of cytotoxicity and considering complex formation and DNase protection studies as well. The SDS-induced release of p5α-Red was also performed on both samples because releasing pDNA on the target side is as an important issue as protecting it from serum nucleases. It was found that SLN:p5α-Red vectors with all applied complex ratios were decomplexed in presence of SDS 1% as seen in Fig. 4 lanes 9–13. Once the optimal SLN:p5α-Red vectors ratio was determined, we proceed to the physicochemical characterization of SLNs and SLN: p5α-Red vectors. DLS measurements were performed in order to investigate the particle size, PDI and zeta potential of SLNs and SLN:p5α-Red (300:1, w/w) vectors. As can be seen in Table 1, the particle size was measured as 18.70 nm for SLNs and 62.65 nm for SLN:p5α-Red (300:1, w/w) vectors. Small size is an important factor to penetrate the cells however, there should be a balance between the size and compact structure. It can be a disadvantage in terms of transfection if the SLN:p5α-Red vectors forms a very tight complex because this may prevent nanoparticles from releasing nucleic acids into the cells. PDI can be used to describe the uniformity of nanoparticles. There is not a certain acceptable limit for PDI, however when the PDI value is lower than 0.4 for DLS measurements, the particles are evaluated as monodisperse. This number can be even higher for biological particles. As seen in Table 1, both SLNs and SLN:p5α-Red (300:1, w/w) vectors can be evaluated as monodisperse particles. Zeta potential is another important point of physicochemical characterization because it is the sign of electrostatic interactions between SLN and p5α-Red. Zeta potential of SLNs are positive and measured as 20.4 (±1.11) mV while SLN:p5α-Red (300:1, w/w) was measured 12.90 (±1.54) mV. The reason for the reduction is due to the effect of negatively charged phosphate chains of nucleic acids to the electrostatic sum up. At SLN: p5α-Red ratio higher than 300:1 (w/w), it is possible to obtain vector systems with higher zeta potential value, due to the increasing amount of cationic lipid. However, an increase in PDI value due to the intensity of particles not participating in the complex and an increase in toxicity due to the amount of cationic lipid content could be observed in that case. SLN:p5α-Red ratio supposed to be determined with various factors. Optimal complex formation determined by gel retardation assay needs to be endorsed with size and zeta analysis. For gene delivery, the positive charge of the vector is an advantage to obtain electrostatic interaction with the negatively charged cell membrane while high zeta potential can cause cytotoxicity. Compact vector formation due to the size and zeta potential is another criteria for the permeation of the particles through the membranes [51].