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  • Sorafenib European Journal of Pharmaceutical Sciences br The release profiles


     European Journal of Pharmaceutical Sciences 127 (2019) 142–150
    The release profiles of the Nile red loaded particles formed by the thin film method were evaluated in vitro using the conventional dialysis bag method (D'Souza, 2014). 0.5 mL of Particles containing Nile red with D:M ratios 1, 2 and 3, were placed in a dialysis bag (cellulose membrane, molecular weight cut-off 14 kDa) containing 2.5 mL 2 mM HEPES buffer. The dialysis bag was placed in a beaker containing 25 mL of 2 mM HEPES buffer (release buffer). The beaker was stirred con-stantly at room temperature and protected from light for 7 days. Sam-ples were taken at time 0, 2, 4, 8 and 24, 48, 86 and 124 h. At these intervals, 400 μL of the release medium was removed and replaced with fresh HEPES buffer. The 400 μL samples were evaporated and re-suspended in a mixture of 1:1 ethanol:HEPES solution and the Nile red concentration was determined by means of fluorescence on a Varioskan LUX Multimode Microplate Reader (Thermo Fischer). All experiments were performed in triplicate.
    2.11. Nile red incorporation
    Nile red incorporation was tested by Sorafenib on different columns. Particles with D:M ratio of 1 were prepared with the thin film method, after rehydration the particle suspension was either passed through a Minisart® 0.2 μm filter (Sartorius), a PD10 desalting column (Sephadex G-25, GE healthcare) or an Amicon Ultra Centrifugal Filter (3 kDa MWC). The filtrate was hereafter evaporated and redissolved in 1:1 EtOH:HEPES. Samples of free Nile red in 1:1 EtOH:HEPES solutions with the same concentrations as in the particles were used as controls. The Nile red concentration before and after filtration was determined by means of fluorescence on a Varioskan LUX Multimode Microplate Reader (Thermo Fischer).
    2.12. Statistical analysis
    Analysis of the data was performed with Microsoft Excel (Version 2016) (WA, USA). All graphs show means and standard deviation, calculated in Excel. Difference between the means of two or more groups was tested using Excel's Students t-Test and ANOVA with Tukey HSD Test performed with R (Version 3.4.0), respectively. Groups with p-value < 0.05 are considered significantly different. We have chosen not to remove any outliers in the datasets, this gives a high standard deviation for some of the samples.
    3. Results
    MDEA/siRNA particles were prepared using the stirring method at different N:P ratios with mismatched (siMM) or GFP targeted (siGFP) siRNA. The particles and unformulated siRNA were then used to transfect GFP expressing H1299 cells (Fig. 1). The GFP knockdown was evaluated using flow cytometry. At N:P = 5, median GFP fluorescence was significantly reduced compared to the control group (45%, p = 1.7 × 10−7). Silencing at N:P = 10 was similar to that of N:P = 5. Lower NP ratios and the controls made with pure siGFP (no MDEA, N:P = 0) did not result in any knockdown. To test if MDEA particles mediate toxicity we performed a resazurin assay on transfected H1299 cells (Fig. 2). The cells were added MDEA alone or MDEA/siRNA particles formed using the stirring method at different N:P ratios in a 96 well plate. The cell viability was reduced by up to 40% by MDEA with and without siRNA. The diameter of MDEA/siRNA particles formed using the stirring method at different N:P ratios was measured using dynamic light scatter (Table 1). Particles form at N:P = 1 and particle size appears to be si-milar at all tested N:P ratios with diameters ranging from 119.1 nm to 161.1 nm. Additionally, the polydispersity index was maintained with an average of 0.238 for NP 1–10 for both siMM and siGFP indicating monodispersed samples. The particle size of below 170 nm is within the
    Fig. 1. GFP silencing induced by MDEA/siRNA particles made using the stirring method. The figure shows the mean normalized GFP fluorescence intensity of H1299 cells 48 h after transfection with MDEA/siRNA containing 50 nM siRNA at different NP ratios. For siRNA, G is siGFP and M is siMM. N:P = 0 corre-sponds to unformulated siRNA.