Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: Three IOs with PEG-PLA were prepared by microfluidic chip to obtain IPM. si HSP47 and si HMGB1 were delivered to treat hepatic fibrosis by targeting activated hepatic stellate cells with the addition of FAPi-PEG-PLA in the formulation. Meanwhile, attenuated ABC phenomenon of IPM was observed.

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: Formulation

Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: a Confocal microscopy images of HSCs co-incubated with IPM/Cy5-si NC formed by different IOs for 2 h, with DAPI labeling nucleus. b Flow cytometry assay demonstrating the highest cellular uptake efficiency of IPM (IOA2, PEG5000-PLA8000) (n = 3 cell culture wells). c The confocal images of the lysosomal escape experiments revealing a decline in the co-localization signals from 4 to 6 h, with LysoTracker Green labeling of lysosomes and Hochest 33342 labeling of nuclei. d Statistical analysis of Pearson’s R for co-localization of Cy5 signal with lysosomal signal. At 4 h, the Pearson’s R in the IPM (IOA2) group was <0.5, changing from moderate to low correlation (n = 3 cell culture wells). e Statistical analysis of Overlap R for co-localization of Cy5 signal with lysosomal signal (n = 3 cell culture wells). f TNS assay for the apparent pKa of the three blank carriers (n = 3 independent experiments). g In vitro RNAi using IPM encapsulating si HSP47 and si HMGB1 formed by PEG5000-PLA8000 and IOA2, IOLA4, IOLA8, with LNP serving as a positive control and LNP/si NC serving as a negative control (n = 3 cell culture wells). The qPCR results showing the effective silencing of the HSP47 ( h ) and HMGB1 ( i ) genes in vitro, with LNP and Lipo2000 serving as positive controls and LNP/si NC and no siRNA serving as negative controls (n = 3 cell culture wells). Data were analyzed by One-way ANOVA followed by Tukey test. Values represent mean ± standard deviation (SD).

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: Confocal Microscopy, Incubation, Labeling, Flow Cytometry, Cell Culture, In Vitro, Positive Control, Negative Control, Standard Deviation

Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: a GPC molecular weight distribution of FAPi-PEG5000-PLA8000 and PEG5000-PLA8000. b qPCR results demonstrating upregulation of FAP mRNA expression in HSCs following bleomycin stimulation (n = 3 cell culture wells). c Flow cytometry analysis of the cellular uptake efficiency of IPM and FAPi-IPM by HSCs before and after bleomycin stimulation. FAPi-IPM exhibits targeting capability, being more effectively internalized by HSCs, with significant differences observed after bleomycin stimulation (n = 3 cell culture wells). d Competitive inhibition assay with varying concentrations of free FAPi, showing dose-dependent cellular uptake of FAPi-IPM (n = 3 cell culture wells). e Confocal microscopy results indicating increased intracellular fluorescence intensity in FAPi-IPM/Cy5-si NC -treated cells after bleomycin stimulation, with a decrease in intracellular fluorescence intensity as free FAPi concentration increases. f Western blot results of IPM/si HMGB1 +si HSP47 and FAPi-IPM/si HMGB1 +si HSP47 after bleomycin stimulation of HSCs, with LNP/si HMGB1 +si HSP47 serving as a positive control. g , h Effective downregulation of target genes HSP47 and HMGB1 relative expression by IPM and FAPi-IPM (n = 3 cell culture wells). i Schematic representation of hepatic fibrosis modeling. j ROI values in the liver of mice with hepatic fibrosis (n = 3 mice). k Flow cytometry showing FAM positivity of activated hepatic stellate cells (α-SMA labeled) in the livers of mice with hepatic fibrosis (n = 3 mice). l The immunofluorescence results of liver tissues from mice with hepatic fibrosis 24 h after the tail vein injection, with DAPI labeling of the nuclei and α-SMA labeling of activated hepatic stellate cells. m Fluorescence imaging of the liver 24 h after injection in mice with liver fibrosis. Data were analyzed by ANOVA followed by Tukey test. Values represent mean ± standard deviation (SD).

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: Molecular Weight, Expressing, Cell Culture, Flow Cytometry, Inhibition, Confocal Microscopy, Fluorescence, Concentration Assay, Western Blot, Positive Control, Labeling, Immunofluorescence, Injection, Imaging, Standard Deviation

Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: a Schematic representation of the dosing regimen for targeting hepatic stellate cells with FAPi-IPM co-delivery of si HSP47 and si HMGB1 for the treatment of hepatic fibrosis (n = 5 mice). b H&E staining results of the liver showing histological changes. c Masson’s trichrome staining results of the liver demonstrating a reduction in collagen deposition area in the FAPi-IPM group. d Quantification of collagen deposition area. e Immunohistochemical staining for HSP47 in the liver showing a decrease in HSP47-positive area and expression in the FAPi-IPM group. f Proportion of HSP47-positive area. g Immunohistochemical staining for HMGB1 in the liver showing a decrease in HMGB1-positive area and expression in the FAPi-IPM group. h Proportion of HMGB1-positive area. i– k Serum levels of TNF-α, AST, and ALT in mice. The FAPi group shows a decrease in ALT and AST levels to the normal range, with no significant difference compared with the negative control group. l Liver appearance showing changes in the PBS group and ‌alleviation in the FAPi-IPM group. m Body weight curve of mice showing weight recovery after administration in the treatment groups. n Western blot results demonstrating inhibition of HSP47 and HMGB1 protein expression in the treatment groups. o , p Significant silencing of HSP47 and HMGB1 protein expression in the FAPi-IPM group in vivo. q , r qPCR results demonstrating significant gene silencing of HSP47 and HMGB1 in the treatment groups. Data were analyzed by ANOVA followed by Tukey’s multiple comparisons test. Values represent mean ± standard deviation (SD).

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: Staining, Immunohistochemical staining, Expressing, Negative Control, Western Blot, Inhibition, In Vivo, Standard Deviation

Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: Design of siRNA sequences for HMGB1 and HSP47

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: Construct, Sequencing

Journal: Nature Communications

Article Title: Ionizable polymeric micelles (IPMs) for efficient siRNA delivery

doi: 10.1038/s41467-024-55721-w

Figure Lengend Snippet: RT-PCR primer sequences

Article Snippet: Primers for qPCR targeting mouse HMGB1 , HSP47 , fibroblast activation protein ( FAP ), and GAPDH were synthesized by Guangzhou RiboBio Co., Ltd (Table ).

Techniques: