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<p>DMPC/2/siRNA treatment of Jurkat T cells, 48 hours from the beginning of transfection.</p

Remote Loading of Aloe Emodin in Gemini-Based Cationic Liposomes

Anthraquinone compound aloe-emodin (AE) has shown antineoplastic, antibacterial, antiviral, and anti-inflammatory properties and scavenging activity on free radicals. Because of these therapeutic features, AE has been attracting increasing interest and could be applied in the curing of many diseases. However, until now the physicochemical features of this compound have not been fully investigated; furthermore, its wide application might be hindered by its scarce solubility in aqueous media (∼19 μM). The inclusion of AE in nanocarriers, such as cationic liposomes, could allow its delivery effectively and selectively to target sites, reducing side effects in the remaining tissues. In this work, the weak acid nature of AE, because of its two phenolic functions, was exploited to load it remotely in the internal aqueous phase of liposomes in response to a difference in pH between the inside and outside of the liposomes, pH_in > pH_out. The inclusion of AE in gemini-based cationic liposomes by the acetate gradient method was obtained at high AE/lipid ratios (up to 1:30)

Dynamic light scattering.

<p>Hydrodynamic diameter distribution functions (averaged by number) obtained by CONTIN analysis of DLS data (scattering angle 90°, T = 25°C) for DMPC/1 liposomes (panel A); DMPC/2 liposomes (panel B); DMPC/1/siRNA lipoplexes (panel C); DMPC/2/siRNA lipoplexes (panel D).</p

Lyp protein expression in Jurkat T cells after siRNA s/a transfection using a commercial system.

<p>(A) siRNA transfection with siRNA having higher affinity to the target mRNA sequence (siRNA1) resulted in a reduction of Lyp O.D. (optical density) percentages to untreated cells to 44%, 43% and 48% with the doses of 40-60-80 pmols respectively after 48 hours of transfection. O.D. values of Lyp and β-actin were obtained with ImageLab software. Lyp O.D. values for every treatment were normalized to the corresponding β-actin values. No reduction was obtained with siRNA molecule with lower affinity (siRNA2). * indicates p<0.05. (B) Representative WB image with all the corresponding experimental groups is shown. Lyp O.D. % with respect to untreated cells’ O.D. values calculated as percentage to untreated cells’ O.D. ‘Untreated’ represents the basal control level (100%) of Lyp protein expression in cells cultured in RPMI. The efficacy of transfection is calculated from the difference between 100 and the test O.D. percentage in each experiment.</p

Circular dichroism spectroscopy.

<p>(A) CD spectra of 1.3 <b>μ</b>M siRNA in buffer solution (5 mM HEPES, 0.1 mM EDTA, pH 7.4). (B) CD spectra of 1.3 <b>μ</b>M siRNA in the DMPC/1 formulation. (C) CD spectra of 1.3 <b>μ</b>M siRNA in the DMPC/2 formulation. Spectra were recorded at 25°C.</p

Confocal microscopy analysis of lipoplexes incorporation in Jurkat T cells.

<p>Data are shown after 60 minutes and 4 and half hours of incubation. Arrows indicate the localization of liposome fluorescence (red spots) compared to cell membrane (green) and nucleus (blue). Z-reconstructions obtained by confocal microscopy show the internalization of lipoplexes in Jurkat T cells in the X- and Y-axis projections. Plasma membrane is stained with wheat germ agglutinin (WGA), whereas nuclei are counterstained with Hoechst. Bar: 10 μm.</p

Lyp protein expression in Jurkat T cells 72 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

<p>Representative duplicate experiment among all replicas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175784#pone.0175784.s004" target="_blank">S4 Fig</a>. (A) Lyp expression in Jurkat T cells after O/N transfection with DMPC/1 alone (DMPC/1), 20 pmols of siRNA alone (siRNA20) and 20 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA20) or cultured in RPMI. 20 pmols of siRNA complexed with DMPC/1 resulted in a 15% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA (siRNA60) complexed with DMPC/1 (DMPC/1/siRNA60). 33% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA (siRNA100) complexed with DMPC/1 (DMPC/1/siRNA100). 45% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown.</p

Evaluation of cell death after lipoplexes internalization.

<p>Flow cytometric analysis of HD PBMC (left) and Jurkat cells (right) treated with rhodamine-marked lipoplexes for 4 and a half hours. The histogram shows both the percentage of ‘transfected’ lymphocytes (rhodamine⁺ cells) and the percentage of dead cells among the transfected ones (DAPI⁺ and rhodamine ⁺ cells).</p

Confocal microscopy analysis of Lyp protein expression in Jurkat T cells after transfection with lipoplexes.

<p>The comparison shown is between samples transfected with 60 and 100 pmols of Lipo/siRNA lipoplexes, and analyzed after 72 hours from the beginning of the O/N transfection. Control cells are untreated and cultured in RPMI (upper panels). Lyp protein expression is revealed by anti-mouse IgG conjugated to Alexa Fluor 555 (red signal). Cell morphology and DNA are detected by WGA (green) and Hoechst (blue) staining, respectively. Bar: 20 μm.</p

Lyp protein levels in Jurkat T cells 48 hours after the beginning of O/N transfection with different doses of siRNA s/a in DMPC/1/siRNA lipoplexes.

<p>Representative duplicate experiment among all replicas in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175784#pone.0175784.s004" target="_blank">S4 Fig</a>. (A) Lyp expression in Jurkat T cells cultured in RPMI or after O/N transfection with DMPC/1, 20 pmols of siRNA and 20 pmols of siRNA in DMPC/1/siRNA lipoplexes (DMPC/1/siRNA20). 20 pmols of siRNA complexed with DMPC/1 resulted in a 31% reduction of Lyp expression. (B) Same experiment as in A using 60 pmols of siRNA complexed with DMPC/1 (DMPC/1/siRNA60). 39% reduction of Lyp expression was obtained. (C) Same experiment as in A using 100 pmols of siRNA complexed with DMPC/1 lipoplexes (DMPC/1/siRNA100). 47% reduction of Lyp expression was obtained. (D) Representative WB image within all experimental groups is shown. Under each blot Lyp O.D. values for every treatment are normalized over the corresponding β-actin values. All percentages were expressed relatively to untransfected cells (RPMI) that is considered the 100% of basal Lyp expression. Graphs A, B, C show the mean values and their standard deviations. In all experimental conditions (<i>vide infra</i>), a decrease in Lyp protein level was not observed in cells treated with the liposome alone or, more importantly, in cells treated with the correspondent dose of the siRNA s/a free molecule.</p