Visualization of the process of a nanocarrier-mediated gene delivery: stabilization, endocytosis and endosomal escape of genes for intracellular spreading

Abstract

Nanoparticles have been widely applied as gene carrier for improving RNA interference (RNAi) efficiency in medical and agricultural fields. However, the mechanism and delivery process of nanoparticle-mediated RNAi is not directly visualized and elucidated. Here we synthesized a star polymer (SPc) consisted of a hydrophilic shell with positively-charged tertiary amine in the side chain, which was taken as an example to investigate the mechanism in gene delivery. The SPc could assemble with dsRNA spontaneously through electrostatic force, hydrogen bond and van der Waals force. Interestingly, the SPc could protect dsRNA from degradation by RNase A and insect hemolymph, thus remarkably increasing the stability of dsRNA. Meanwhile, the SPc could efficiently promote the cellular uptake and endosomal escape for intracellular spreading of dsRNA. Transcriptome analysis revealed that the SPc could up-regulate some key genes such as Chc, AP2S1 and Arf1 for activating clathrin-mediated endocytosis. Furthermore, the suppression of endocytosis hindered the cellular uptake of SPc-delivered dsRNA in vitro, and the subsequent RNAi effect was also disappeared in vivo. To our knowledge, our study is the first direct visualization of the detailed cellular delivery process and mechanism of nanocarrier-mediated gene delivery. Above mechanism supports the application of nanocarrier-based RNAi in gene therapy and pest management.

Keywords: Cellular uptake; Clathrin; Endocytosis; Nanocarrier; RNA interference; dsRNA.

Fig. 1

Self-assembly mechanism of dsRNA/SPc complex. a Gel electrophoresis assay of dseGFP retardation by SPc. One μg dseGFP was mixed with SPc at various mass ratios, and the mixture (10 μL) was incubated and then analyzed. M: DNA marker. b ITC titration of dseGFP (0.333 μM) into SPc solution (5.1 μM). The heats of interaction during each injection were calculated by the integration of each titration peak. The test temperature was 25 °C

Fig. 2

Enhanced stability of SPc-complexed dsRNA. a The dseGFP degradation by RNase A. One μg dseGFP was added with RNase A to prepare the reaction solution (dseGFP: 100 ng/μL), and the mixture was incubated for 20 min at 37 °C. M: DNA marker. Gel electrophoresis assay (b) and relative band density (c) of SPc-complexed dseGFP treated with RNase A. The RNase A was used to treat dseGFP/SPc complex (dseGFP: 1 μg). Then the dseGFP/SPc complex was decomplexed in 0.3% SDS solution. Each treatment was repeated 3 times. d Relative dsRNA amount of SPc-complexed dseGFP treated with RNase A. The decomplexed dseGFP was purified and quantified. Each treatment was repeated 3 times. Statistical analysis was conducted using independent t-test at the P = 0.05 level of significance. e–h’ Fluorescent images of immune cells treated with naked dseGFP (e-e’) or SPc-complexed dseGFP (f–h’). The dseGFP and dseGFP/SPc complex were incubated with hemolymph for 3 h (dseGFP: 500 ng). Blue: DAPI. Green: dseGFP. g-g’ Plasmatocyte. h–h’ Granulocyte

Fig. 3

Improved endocytosis and exocytosis of dsRNA/SPc complex. (a-b) Cellular uptake of naked dseGFP (a) and dseGFP/SPc complex (b). The cells were incubated with dseGFP and dseGFP/SPc complex for 6 h, respectively (dseGFP: 500 ng). Blue: DAPI. Green: dseGFP. (c–e’) Vesicle release of SPc-delivered dseGFP into cytoplasm. The cells were incubated with dseGFP and dseGFP/SPc complex, respectively (dseGFP: 500 ng) for 6 h. A surface plot test was conducted using ImageJ. Three typical regions were marked. f, g’ Endosomal escape of SPc-delivered dsRNA. The cells incubated with SPc-delivered dsRNA for 6 h were imaged. Red: Rab7 marking the late endosome. h–h’ Potential exocytosis of dsRNA/SPc complex. The cells incubated with SPc-delivered dsRNA for 6 h were re-suspended in fresh medium, and then imaged. i Schematic illustration of endocytosis and exocytosis of SPc-delivered dsRNA

Fig. 4

Comparative transcriptome analysis of Sf9 cells treated with naked dseGFP and dseGFP/SPc complex. Analysis of differentially expressed genes (DEGs) with volcano plot (a) and GO annotation (b). Cells were collected at 6 h after incubation with dseGFP and dseGFP/SPc complex (dseGFP: 500 ng) for RNA extraction. c Heatmap of the endocytic pathway. d QRT-PCR analysis of the genes related to clathrin-mediated endocytosis. The expression levels of AP2S1, Arf and Chc were determined at various time points after the treatment of dseGFP or dseGFP/SPc complex (dseGFP: 500 ng). Each treatment consisted of 3 replications. The actin and ribosomal protein S15 (RPS-15) were used as reference genes. Asterisk indicates significant difference in gene expression between dseGFP/SPc complex and naked dseGFP treatment according to independent t-test (ns.: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001)

Fig. 5

Pharmacological inhibitor blocks the SPc-mediated dsRNA delivery. a, b’ Inhibitor application hindered the cellular uptake of dseGFP/SPc complex. The cells were exposed to 0.2 μM Bafilomycin-A (inhibitor) for 30 min, incubated with dseGFP/SPc complex, and then examined. Blue: DAPI. Green: dseGFP. c Schematic diagram of pharmacological assay toward S. frugiperda. The 2-day-old larvae were fed with Baf A for 2 days. Then, RNAi was performed using 4-d-old larvae. d QRT-PCR analysis of ATP-d gene expression in larvae fed with inhibitor. Each treatment consisted of 3 replications. Different letters on columns indicate significant differences (Tukey HSD test, P < 0.05)