Artificial Control of Gene Silencing Activity Based on siRNA Conjugation with Polymeric Molecule Having Coil–Globule Transition Behavior

A new strategy for controlling gene silencing activity of siRNA in the cell was developed in the present study. siRNA was linearly conjugated with PNIPAAm, where coil–globule transition of the conjugated PNIPAAm allows thermoresponsive exposure of the vicinal siRNA molecule; a coil form of PNIPAAm (<i>T</i> < LCST) inhibits siRNA interaction with gene silencing-related proteins due to the steric hindrance effect, while a globule form of PNIPAAm (<i>T</i> > LCST) allows a ready access of siRNA to gene silencing pathway. As a result, at <i>T</i> > LCST, PNIPAAm-siRNA elicited effective association of siRNA with a gene silencing-related protein of Ago2, while siRNA recruitment into the gene silencing pathway was significantly suppressed at <i>T</i> < LCST. Ultimately, gene silencing efficacy of PNIPAAm-siRNA was close to unconjugated siRNA at <i>T</i> > LCST (∼80%), while it was dramatically decreased to ∼20% at <i>T</i> < LCST, suggesting that coil–globule transition of the conjugated polymer can control the bioactivity of the vicinal siRNA molecule

Time courses of monocyte subsets regarding stroke complications.

<p>Classical and intermediate Mo were increased, while non-classical Mo were decreased. Patients were divided into (<b>A</b>) with or without PI, (<b>B</b>) excluding SAI, (<b>C</b>) and with or without SAI. *p<0.05, t test, compared to control. †p<0.05, t test, compared to other group in category. PI = progressing infarction, SAI = stroke-associated infection.</p

Stroke subtype and monocyte subsets from day 0-2 (hyperacute phase).

<p>(<b>A</b>) The overall monocyte counts in the LAA and CE groups were significantly increased as compared to the control. (<b>B</b>, <b>C</b>, <b>D</b>) The percentages for the monocyte subsets in the CE group were significantly changed as compared to the control. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. †p<0.05, ANOVA, compared to CE. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.</p

Time courses of monocyte subsets after stroke.

<p>(<b>A</b>) The overall number of circulating monocytes was increased. The percentages of (<b>B</b>) classical and (<b>C</b>) intermediate Mo were increased, whereas that of (<b>D</b>) non-classical Mo was decreased. Bars show the mean ± SEM. *p<0.05, t-test, compared to control. Control, n=24; day 0-2, n=36; day 3-7, n=35; day 12-16; n=33; day of hospital discharge, n=26.</p

Analysis of monocyte subsets.

<p>(<b>A</b>) Monocytes were gated in a circle using forward scatter (FSC) and side scatter (SSC) plots. (<b>B</b>) Classical monocytes were identified by high expression of CD14 and no expression of CD16 (CD14^highCD16^-), intermediate monocytes by high expression of CD14 and various levels of positivity for the CD16 molecule (CD14^highCD16⁺), and non-classical monocytes by scant expression of CD14 and high expression of CD16 (CD14 ^dimCD16^high). The proportions of each subset were evaluated by comparing the number of cells (dots) in the individual compartment to the total number of monocytes enclosed in the circle designated as P1.</p

Stroke subtypes and intermediate monocytes after hyperacute phase of stroke.

<p>(<b>A</b>–<b>C</b>) A significantly elevated percentage of intermediate Mo in the CE group persisted until the day of discharge. *p<0.05, t-test, as compared to control. C: control; LAA: large artery atherosclerosis; CE: cardioembolism; SAO: small artery occlusion.</p

Dynamics of pro-inflammatory cytokines after stroke.

<p>(<b>A</b>) In the SAI group (closed diamond), serum IL-6 levels were elevated from 0–7 days after stroke, (<b>B</b>) while serum IL-17A levels were elevated in the PI group without SAI (closed square) from 3–7 days after stroke.</p

Minimal data for the second survey.

(XLSX)</p

Diagnostic criteria for MG.

Diagnostic criteria for MG.</p

Onset age by categories.

Box plot and violin plot of onset age by categories. The horizontal line within the box represents the median value. The box ends to define the 25th and 75th quantiles, expressed as the first and third quartiles. The lines that extend from the box end are whiskers. The whiskers extend from the ends of the box to the outermost data point that falls within these distances. 1st quartile–1.5 x (interquartile range), 3rd quartile+1.5 x (interquartile range). For statistical analysis, Steel-Dwass All Pairwise Comparison was performed.</p