Materials
[2-(Methacryloyloxy)ethyl] trimethylammonium chloride solution (QDMAEMA, 80 wt% in H2O), N,N-dimethylacrylamide (DMA, 99%), Dulbecco’s Modified Eagle’s Medium (DMEM), sodium chloride (NaCl, 99.5%), D2O (99.9%), ethanol (100%), hydrochloric acid (HCl, 12 M) and 10X phosphate-buffered saline (PBS) were purchased from Sigma Aldrich. 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyano-pentanoic acid (CCCP, 95%) was purchased from Boron Molecular. 4,4’-Azobis(4-cyanovaleric acid) (ACVA, 97%) was purchased from Acros Organics. Vha26 (V-ATPase) 222 bp dsRNA was synthesised by Genolution AgroRNA (4.68 µg µL-1), with the nucleotide sequence specific to the pest insect, Drosophila suzukii (Taning et al. 2016). Regenerated cellulose dialysis membrane (3,500 g mol-1 MWCO) was purchased from Fisher Scientific. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (CellTiter 96® AQueous One Solution Cell Proliferation Assay) was purchased from Promega. ProLong™ Gold Antifade Mountant with DAPI was purchased from Thermo Fisher Scientific. Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit Cy3 was purchased from MirusBio. Ultrapure Milli-Q water (resistivity of minimum 18.2 MΩ.cm) was used for synthesis, solution preparation and dialysis, and nuclease-free water (VWR) was used for biological assays.
Polymer synthesis
Synthesis of homopolymer (Q, macro-chain transfer agent (CTA)) and diblock copolymers was conducted as described in a previous publication (Pugsley et al. 2022), but is mentioned in brief below.
Hydrophilic homopolymer: Poly[2-(methacryloyloxy)ethyl] trimethylammonium chloride (Q) was synthesised by aqueous RAFT polymerisation as a macro-CTA. Quaternised monomer (QDMAEMA) was stirred at 70 °C for 1.5 h before quenching by exposure to air. The Q macro-CTA, with a degree of polymerisation of 110, was purified by dialysis in Milli-Q water (< 3500 g mol-1) and lyophilised.
Hydrophilic diblock copolymers: Q macro-CTA was chain extended with poly(N,N-dimethyl acrylamide) (D) (to measured degrees of polymerisation of 57, 89 and 219) in aqueous solution at 70 °C for 4 h prior to dialysis in Milli-Q water (< 3500 g mol-1) and lyophilisation.
Polymer characterisation
The polymers were characterised by 1H NMR spectroscopy (400 MHz) to confirm degree of polymerisation (DP) and molecular weight (Mn), and by aqueous gel permeation chromatography (aq-GPC) to confirm dispersity (Đ) (Agilent 1260 Infinity 2, with two PL aquagel-OH Mixed-H columns, eluent 0.8 M NaNO3, 0.01 M NaH2PO4, 0.05 wt% NaN3 in Milli-Q water, adjusted to pH 3 using 37% HCl). These data can be found in our previously published work (Pugsley et al. 2022).
Ex vivo degradation
Intestines were dissected from 3rd instar D. suzukii larvae (L3), after 2 h starvation to clear the gut of food, and homogenised in Milli-Q water using a plastic homogeniser (30 x intestines / 100 μL). For adults, male and female flies were starved for 2 h, and the intestines were then removed from flies of mixed sex and homogenised in Milli-Q water (7 x intestines / 100 μL). Subsequently, all homogenates were centrifuged at 13,000× g for 20 min and the supernatant collected and stored at −20 °C until required.
Pre-determined aliquots of Q110, Q110-b-D57, Q110-b-D89 or Q110-b-D219 polymers were added to 1 μg dsRNA to provide a N/P ratio = 2.5. Solutions were incubated at RT for 1.5 h, and naked dsRNA and nuclease-free water solutions were prepared. 7.1 µL of adult gut solution or 1.6 µL of L3 gut solution (equivalent of ½ a gut was used per 1 μg dsRNA (i.e. per gel lane), which was sufficient to fully degrade naked dsRNA (Song et al. 2017; Chen et al. 2021)) were added to samples of naked dsRNA and polyplex solutions. Following this, the samples were incubated for 30 min or 24 h, respectively, at 26 °C, 60 % relative humidity (RH). Each solution was loaded (with 2 µL 6X blue/orange loading dye) onto a 2% (wt/vol) agarose gel stained with ethidium bromide (EB, 20 ng mL-1), prepared with 1X TAE (Tris base, acetic acid and EDTA) buffer. Assays were run for 25 min at 90 V. A 100 bp DNA ladder (New England Biolabs, UK) was run for comparison. Gels were imaged under a UV transilluminator at 365 nm.
Cell culture
HEK-293T cells were cultured in 75 cm2 flasks in a 37 °C, 5% CO2 and 95 %RH incubator. Media was Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS (vol/vol) and 1% (wt/vol) penicillin-streptomycin. Cell confluence/density was monitored and cells were passaged as required.
Cy3-labelling of dsRNA
DsRNA (vha26, 222 bp) was labelled with Cy3 labelling kit fluorophore (MirusBio) according to kit protocols and purified by precipitation in ethanol and stored at -20 °C until required.
Flow cytometry
A 12-well plate of HEK-293T cells were seeded at 0.1 x 106 cells/well density, 24 h before treatment. Polyplexes were prepared 24 h prior to treatment through addition of 20 μL Cy3-labeled dsRNA (50 ng μL-1) to 20 μL polymer (concentrations varied to provide a N/P ratio = 5). Polyplexes, Cy3-labelled dsRNA and polymer solutions were individually applied to HEK-293T cell wells. Nuclease-free water was applied in the absence of either polymer or dsRNA, and a nuclease-free water control was conducted. After 4 h, cells were placed on ice and then lifted from wells by physical disturbance. Cells were pelleted by centrifuge, and media/treatment was removed before washing and resuspension of cells in fresh, ice-cold DMEM media. Cy3-positive cells were analysed by flow cytometry using a Cytoflex S (Beckman Coulter) with filters set at 561 nm excitation and 585 nm emission wavelegnths. Data was analysed using FlowJo software. Examples of gating and dead cell determination by 7-AAD is presented in Fig. S1 in the SI.
Confocal microscopy
Solution preparation and cell seeding were performed as described above. However, cells were cultured on microscope slides, introduced to the 12-well plate, and after incubation the cells were fixed with 4% (wt/vol) paraformaldehyde. A DAPI-stain liquid mountant was used to prepare slides for cell imaging on a confocal microscope (Zeiss LSM700).
Cell viability
HEK-293T cells were seeded onto 96-well plates 2 days prior to assay, at a 20,000 cells/well density. A range of polymer concentrations were prepared in Milli-Q water (0.01 – 10 mg mL-1). Aliquots of polymer (100 μL/well) at each concentration were added to cells with 3 replicates, and incubated for 24 h at 37 °C, 5% CO2 and 95 %RH. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, 20 μL) was then added to each well. Cells were incubated with the MTS for 4 h, prior to analysis using a plate reader set at a wavelength of 495 nm. Cells treated with media only were normalised to 100% cell viability and other treatments were normalised with respect to the media cells.
In vivo oral feeding
1st/2nd instar (L1/2) D. suzukii and D. melanogaster larvae were starved on moist filter paper for 3 h prior to addition to artificial diet. Vha26 dsRNA (4.68 g L-1, 8 μL) was complexed with Q110, Q110-b-D57, Q110-b-D99, or Q110-b-D219 (8 μL) at concentrations to provide N/P ratio = 5 and incubated for 1.5 h. Polyplex (16 μL), dsRNA (8 μL) + nuclease-free water (8 μL), polymer (8 μL) + nuclease-free water (8 μL), or nuclease-free water (16 μL) solutions were added to 50 mg of diet and mixed thoroughly for an even distribution. Five starved L1/2 larvae were moved to each 50 mg of treated diet, n = 25 total, and fed for 24 h. Larvae were then removed to fresh diet and monitored for the next 15 days for mortality, assessed by the failure to reach the pupal stage and the absence of any live larvae in or on the surface of the food. Survival results were averaged across assay repeats and normalised against nuclease-free water-fed larvae. Standard deviation was calculated and represented as error bars. Statistical significance was determined using binary logistic regression, performed in SPSS Statistics 26.