Polymer Nanocarrier System for Endosome Escape and Timed Release of siRNA with Complete Gene Silencing and Cell Death in Cancer Cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA

Additional file 1: Figure S1. of Gamma tocotrienol targets tyrosine phosphatase SHP2 in mammospheres resulting in cell death through RAS/ERK pathway

Characterization of sphere cell forming cells cultured from colon cancer cell line HCT-116. Figure S2. Self-renewal gene expressions of Îł-T3 treated HeLa spherical cells. (DOCX 167 kb

Metabolic effects of HCHF diet feeding.

<p>(A) Body weight of Wistar rats on CD or HCHF diet (n = 8). (B) Dorsal view of the rats showing the changes in the total abdominal length caused by the two diets after 16 weeks. ELISA analysis of pro-inflammatory (C) and (D) or anti-inflammatory (E) cytokines in serum (n = 6). Data were analyzed by two-tailed Student’s t test. All data are presented as mean ± SD. P < 0.05 (CD vs HCHF at two time point- week 8 and week 16) was considered to be significant. * = <i>p</i> <0.05.</p

Additional file 1: Table S1. of Characterization of nano-structural and nano-mechanical properties of osteoarthritic subchondral bone

EDS data of subchondral bone plate. Values (mean ± SD) with different superscript letters (a vs b vs c) were significant difference (one-way ANOVA analysis and SNK-q test, P < 0.05). Table S2. EDS data of trabecular bone. Values (mean ± SD) with different superscript letters (a vs b vs c) were significant difference (one-way ANOVA analysis and SNK-q test, P < 0.05). Table S3. The EDS data of mineral crystals are shown as follow. Values (mean ± SD). Significance = P < 0.05. Table S4. The splitting factor data of mineral crystals are shown as follow. Values (mean ± SD). Significance = P < 0.05. (DOC 104 kb

Synovial fluid of 16-week HCHF rats alters macrophage polarization and chondrocyte differentiation in vitro.

<p>Relative qPCR analysis of pro-inflammatory M1-like (A) or anti-inflammatory M2-like (B) genes in BMDMs after synovial fluid stimulation. ELISA analysis of pro-inflammatory (C) or anti-inflammatory (D) cytokines in conditioned medium. (E) Relative qPCR analysis of MMP13, ADAMTS5, COL10, ACAN and SOX9 in micromass cultured ACCs after 7 days of synovial fluid stimulation. (F) GAG release in supernatant of ACCs after synovial fluid stimulation at day 3 and day 7. Data were analyzed by two-tailed Student’s <i>t</i> test. All data are presented as mean ± SD, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5 independent samples.</p

M1 macrophage negatively affects chondrogenic differentiation of chondrocytes.

<p>Relative qPCR analysis of pro-inflammatory M1-like (A) or anti-inflammatory M2-like (B) genes in CD14+ macrophages after cytokine treatment. ELISA analysis of pro-inflammatory (C) or anti-inflammatory (D) cytokines in conditioned medium. Relative qPCR analysis of MMP2 (E), MMP13 (F), RUNX2 (G), ADAMTS5 (H), SOX9 (I), and ACAN (J) mRNA levels in chondrocytes treated with M1 CM or M2 CM. (K) GAG release in supernatant of chondrocytes treated with 50% M1/M2 CM treatment for 3 and 7 days. Data were analyzed by two-tailed Student’s <i>t</i> test. Values represent the mean ± SD of experimental triplicates, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5 independent samples.</p

Dietary intake and body composition in CD and HCHF rats.

<p>Dietary intake and body composition in CD and HCHF rats.</p

Macrophage-like cells increase in inflamed synovium of 16-week HCHF rats and are predominately iNOS+.

<p>(A) Representative immunohistochemical and immunofluorescence analyses of synovial tissues from CD or HCHF diet rats with anti-CD68, anti-iNOS or anti-Arg1. (B) Quantitative assessment of CD68+, iNOS+ or Arg1+ cells of inflamed synovium. Total positive cells per 100 cells were used as a standard measure to quantify. (C, D) qPCR analysis of pro-inflammatory M1-like (C) or anti-inflammatory M2-like (D) genes in synovium after diet stimulation. Data were analyzed by two-tailed Student’s <i>t</i> test. All data are presented as mean ± SD, <i>P</i> < 0.05 was considered to be significant. * = <i>p</i> <0.05. n = 5. Scale bar = 20 μM.</p

Time-dependent histopathologic changes in the joint of HCHF diet-fed rats (8 and 16 weeks).

<p>Histological evaluation of the knee joints. Tissues were stained with safranin-O and fast green (A-B) to estimate the proteoglycan loss among the two time points in HCHF and CD groups. Scale bar = 20 μM. Extent of articular cartilage degradation was graded using Mankin scoring system (E). Safranin-O and fast green staining shows the difference in thickness of the synovial membrane following, (C)8, and (D)16-week HCHF diet Histological scoring showed increased synovial thickening in 8 and 16-week-diet rats (F). Data were analyzed by Mann-Whitney test. All data are presented as mean ± SD. <i>P</i> < 0.05 (HCHF at week 8 vs HCHF at week 16) was considered to be significant. * = <i>p</i> <0.05. n = 8 per each group at each collection time point. Scale bar = 20 μM.</p