Considering the physicochemical stability results on Fig it
Considering the physicochemical stability results on Fig. 5, it was observed that the particles are stable when they are stored at 4 °C for 30 days. After day 30, a significant increase was observed in particle size, but at the end of the 90 days the SLN particle size is still below 100 nm. Stability of the particles could be increased by using lyophilization with cryoprotectants or by reducing the amount of free micelles in the SLN suspension . Since high amounts of genetic material are required to be consumed, experiments using nanoparticles and gene distribution systems must be carried out in small volumes. To examine the in vitro permeation of SLN:p5α-Red vectors in such volumes, a suitable apparatus was designed that allows the evaluation of dialysis in a very small donor volumes. 14000 kDa funny hat membrane was used to mimic topical penetration of vectors. Agarose gel electrophoresis was applied to the residual SLN: p5α-Red vectors in the donor compartment which were previously decomplexed in the presence of SDS 1%. As seen in gel image in Fig. 6, the amount of residual DNA decreases linearly with time. Band densities were analyzed by quantified depending on the band area and the integrity of the bands. Dialyzed volume percentage of the SLN: p5α-Red vector through the membrane was calculated and it was found that nanoparticles are able to pass the membrane and %80.2 of the SLN:p5α-Red vectors were dialyzed after 6 h. Prior to transfection, the cytotoxicity assay was examined to determine the viability of cells. As seen in Fig. 7, cell viability is reduced in relative order against increasing SLNs and SLN:p5α-Red vector doses. One of the main causes of toxicity in cationic SLN formulations is zeta potential. SLNs have higher zeta potential than the SLN:p5α-Red vectors and show relatively higher cytotoxicity in parallel. SLN:p5α-Red vector doses showing cell viability above 70% were accepted as viable reference doses for transfection studies. Therefore, no significant cytotoxicity was observed in the concentration range of 22.5–90 μg/well for SLN:p5α-Red vectors. Transfection ability is one of the key parameters that supposed to be determined by gene silencing assays. One of the easiest and most convenient methods of monitoring transfection is the expression of genes encoding reporter proteins like fluorescent biomarkers . Transfection ability of developed SLN was shown by using the pEGFP vector. In this study, the cells were transfected by SLN:pEGFP-C1 vector and EGFP expression was observed under fluorescence microscope (Fig. 8). Since there is no existing transfection protocol for this novel formulation that we developed, this experiment is also a necessary step for the optimization of the transfection protocol. As can be seen in Fig. 8, it was observed that the developed SLN: pEGFP-C1 vectors have the ability to transfect DU-145 cells. Transfection was followed for 24 and 48 h and EGFP expression was observed in more cells after 48 h. This can be used as a preliminary information for transfection with SLN:p5α-Red vectors, which allows shRNA formation during gene silencing. Transfection was then repeated according to the determined protocol using the SLN:p5α-Red vectors. Forty-eight hours after transfection, the amount of p5α-Red enzyme was analyzed by western blot method. Fig. 9, shows the membrane image obtained after western blot analysis. The SLN carrying p5α-Red-C plasmid was used as a negative control in this assay. When compared to control, we analyzed that after 48 h, the western blot band density of 5α-Red enzyme reduced about 65.1% compared to the control. Western blot analysis showed that SLN:p5α-Red (300:1, w/w) vectors were able to silence p5α-Red enzyme synthesis in DU-145 cell line (Fig. 9). In the further studies, it is planned to carry out in vitro analysis of gene silencing effect of developed system against androgenic alopecia for topical application by using Hair Follicle Dermal Papilla Cells (HFDPC) primer cell culture. In addition, the use of a target agent against a prostate cancer treatment can be explored as a safe alternative to currently available DHT inhibitors.