Chemicals
The SPc and PDI-cored cationic dendrimer G2 were synthesized according to the methods described by Li et al.49 and Xu et al.60, respectively. For SPc synthesis, the star initiator Pt-Br was synthesized using pentaerythritol, and the polymerization of star initiator with DMAEMA was then carried out. For G2 synthesis, the synthesis strategy was started from amine-functionalized PDI. The four primary amines selectively reacted with the acrylate group in 2-methacryloyloxyethyl acrylate to prepare the intermediate product, and the methacrylates at the periphery reacted with the thiol group in cysteamine to further prepare the G2.
Cell Culture and Insect Rearing
The Sf9 cells from ovaries of fall armyworms (S. frugiperda) were cultured in Sf-900 II SFM Medium (Gibco, USA) at 27°C supplemented with 10% fetal bovine serum, penicillin (100 unit/mL) and streptomycin (100 μg/mL). Larvae of S. frugiperda were fed on an artificial diet bought from Tuidongzhe Biotechnology Co. (China), and reared under a photoperiod of 16 h light: 8 h dark at 25°C.
Gel Retardation Test and Isothermal Titration Calorimetry (ITC) Assays
The enhanced green fluorescent protein gene (eGFP) was selected for dsRNA synthesis using the T7 RiboMAX expression (Promega, USA). All primers (Table S1) were synthesized by Tsingke (China). A gel retardation test was firstly performed to determine the best mass ratio for the combination of dsRNA with nanoparticle. One μg dseGFP was mixed with SPc or G2 at various mass ratios, and the mixture (10 μL) was incubated at room temperature for 15 min and then analyzed by agarose gel electrophoresis. To determine the interaction of dsRNA with SPc, the 5.1 μM SPc was titrated with 0.333 μM dseGFP in Nano ITC (TA Instruments Waters, USA). The heats of interaction during each injection were calculated by the integration of each titration peak via the Origin7 software (OriginLab Co., USA). The test temperature was 25°C, and the ΔG was calculated using the formula of ΔG=ΔH−TΔS.
Stability Test of Nanocarrier-Complexed dsRNA
To determine the stability of dsRNA, one μg dseGFP was added with RNase A (Sigma-Aldrich, USA) to prepare the reaction solution (dseGFP: 100 ng/μL), and the mixture was incubated for 20 min at 37°C. The hemolymph collected from 5th instar larvae of S. frugiperda82 was diluted with 1×PBS and mixed with 1 μg dseGFP, and the mixture was incubated for 1.5 h at room temperature. The agarose gel electrophoresis was used to detect the integrity of dsRNA treated with RNase A or hemolymph.
To investigate the stability of nanocarrier-complexed dsRNA, dsRNA was mixed with SPc and G2 at the best mass ratio, respectively. The RNase A was used to treat dseGFP/SPc or dseGFP/G2 complex (dseGFP: 1 μg). For the test of SPc-complexed dsRNA, the dseGFP/SPc complex was decomplexed in 0.3% SDS solution and analyzed using agarose gel electrophoresis. The relative band density was determined using ImageJ 1.48v (National Institutes of Health, USA). Meanwhile, decomplexed dseGFP was purified using MEGAclear Kit (Thermo Fisher Scientific, USA), and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Naked dseGFP was applied as control, and each treatment was repeated 3 times. The stability test of G2-complexed dsRNA was performed using the method described by Liu et al.47 Changes in absorption spectra of dseGFP/G2 complex treated with RNase A were detected using NanoDrop 2000 spectrophotometer. The stability of dseGFP/SPc or dseGFP/G2 complex treated with hemolymph was analyzed according to the method of dseGFP/SPc complex treated with RNase A.
For the stability test of nanocarrier-complexed dsRNA in confrontation with immune cells, 100 μL of above hemolymph was added with 400 μL Sf-900 II SFM Medium. Fluorescent dseGFP was synthesized,63 and then mixed with SPc and G2 at the best mass ratio, respectively. Changes in fluorescence intensity of dseGFP complexed with SPc or G2 were then tested using NanoDrop 2000 spectrophotometer. The complexation of dseGFP with G2 influenced the fluorescence intensity of dseGFP, and therefore SPc was used in the following experiments. The dseGFP and dseGFP/SPc complex were incubated with hemolymph for 3 h (dseGFP: 500 ng). The cells were washed, fixed with 4% paraformaldehyde, deposited on the slides using an Antifade Mounting Medium with DAPI (Vector Laboratories, USA), and then examined using a confocal microscope (Leica SP8, Germany).
Cellular Uptake and Intracellular Trafficking of SPc-Delivered dsRNA/siRNA
Fluorescent dseGFP and sieGFP (GenePharma Co., China) were used to determine the cellular uptake of SPc-delivered dsRNA/siRNA. The cells were treated with dseGFP, dseGFP/SPc complex, sieGFP and sieGFP/SPc complex, respectively (dseGFP: 500 ng; sieGFP: 280 ng). Cells were imaged using a confocal microscope at various time points after incubation. The time-lapse imaging for tracing dseGFP/SPc complex was conducted using an inverted fluorescence microscope (Olympus, Japan) after 1 h of incubation, followed by a qualification of the intracellular fluorescence intensity by Surface Plot Program using ImageJ 1.48v at various time points.
To investigate the intracellular trafficking of SPc-delivered dsRNA, cells incubated for 6 h were re-suspended in fresh medium for 30 min, and then imaged using an inverted fluorescence microscope. The intracellular fluorescence intensity was determined using Surface Plot Program similarly. Meanwhile, above cells were deposited on the slides, fixed with 4% paraformaldehyde, treated with 0.1% Triton X-100, and then blocked with 0.2% bovine serum albumin. Blocked samples were incubated with primary antibody mouse anti-Rab 7 (1:200, DSHB, USA) overnight, incubated with secondary antibody goat anti-mouse Cy5 (1:200, Jackson, USA) for 1.5 h, and then examined using a confocal microscope.
Transcriptome Analysis
Cells were collected at 6 h after incubation with dseGFP and dseGFP/SPc complex (dseGFP: 500 ng), respectively. Total RNA was extracted from three biological replicates using the TRNzol formulation (TIANGEN, China). The transcript libraries were constructed via Illumina HiSeq sequencing platform. Raw reads containing connectors and with low-quality (Q≤10) were removed. The resulting clean reads were assembled using Trinity software.83 TopHat2 was used to achieve the sequence alignment of clean reads with the reference genome (http://www.insect-genome.com/Sfru/).84 BLASTX was used to compare unigene sequences with the Non-Redundant protein sequence database (NR) and Swiss-Prot database for annotation of unigenes. The expression level of each transcript was presented by FPKM value. Deseq was applied for differential expression analysis between transcripts, and fold change≥1.5 and FDR<0.05 were screen conditions.85
The expression levels of endocytosis-related gene AP2S1, Arf and Chc were determined using quantitative real-time PCR (qRT-PCR) at various time points after the treatment of dseGFP or dseGFP/SPc complex (dseGFP: 500 ng). All primers for qRT-PCR were shown in Table S2. The qRT-PCR was performed with Step One Plus Real-Time PCR system (Applied Biosystems, USA) using Power SYBR® Green Master Mix (Applied Biosystems). The actin and ribosomal protein S15 (RPS-15) were selected as reference genes, and the relative mRNA levels of target genes were normalized to the abundance of two genes using the 2-∆∆CT methods.86
Pharmacological Inhibitors of SPc-Delivered dsRNA
Bafilomycin-A (Baf A) (MCE, USA) was used to inhibit clathrin-mediated endocytosis to determine the major pathway for SPc-mediated cellular uptake.65 For in vitro test, the cells were exposed to 0.2 μM Baf A (MCE, USA) for 30 min, treated with fluorescent dseGFP/SPc complex, deposited on the slides using an Antifade Mounting Medium with DAPI, and then examined using a confocal microscope. For in vivo test, the V-type proton ATPase subunit d (ATP-d) was selected as a target gene, and the dsATP-d was mixed with SPc and 1% volume of surfactant Alkyl Polyglucoside (Wanhua, China) to prepare the dsATP-d/SPc complex formulation (dsRNA and SPc: both 1 μg/μL). The 2-day-old larvae of S. frugiperda were fed with Baf A for 2 d, and the 0.5 μL formulation was applied directly on the notum of 4-day-old larvae. The relative mRNA level of ATP-d was evaluated using qRT-PCR at 24 h after the topical application. Each treatment consisted of 3 replications.
Statistical Analysis
The ANOVA with Tukey HSD test was conducted using SPSS 20.0 (SPSS Inc., USA) at the P=0.05 level of significance. A two-tailed Student’s t-test was performed using Prism 7.0 (GraphPad Software Inc., USA) at the P=0.05 level of significance. The descriptive statistics were shown as the mean value and standard errors of the mean.