Nucleophile responsive charge-reversing polycations for pDNA transfection

Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (∼10×) and the thiol glutathione (∼1000×). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.

Radical polymerisation conversion (ρ) was calculated by monitoring reduction in the 1 H NMR integrals of the monomer unsaturated protons (∫M: 5.6 -6.7 ppm for DMA, 5.5 -6.7 ppm for 4VP) and aromatic protons in case of 4VP (7.5 ppm) relative to the internal standard DSS (0 ppm,

Eq. S1
For a polymerisation containing z monomers, M n,NMR was calculated according to Equation S2.
Here [M x ] 0 is the initial concentration of monomer x, [CTA] 0 is the initial chain transfer agent (CTA) concentration and M Mx and M CTA are the monomer x and CTA molecular weights, respectively.

Eq. S2
Additionaly, the polycationic derivatives of 1 and 2 were found to have strong interactions with the GPC column, thus M n,GPC for the polycations was unable to be measured.

Fluorescently labelled polycations
1 and 2 were labelled with coumarin as a fluorescent tracer to allow for investigation into polymer cellular internalisation of their cationic derivatives. These were prepared using a one-pot aminolysis/thiol-ene procedure according to Scheme S1. Scheme S1. Preparation of coumarin labelled polycations. Scheme is simplified for clarity, with non-functionalised and side product species not shown. 1 coum . 1 (130 mg, 11 μmol pyridine units) was dissolved in 1.5 mL THF and 0.25 mL DMF. The yellow solution was then deoxygenated by argon bubbling for 5 minutes. Ethylamine (120 μL of 2 M solution in THF, 212 μmol) and P(OEt 3 ) (20 μL, 120 μmol) were added, resulting in a solution colour change from yellow to brown. After stirring for 2.5 h at RT, Coumarin maleimide (11.2 mg, 29 μmol) was added and the solution was left stirring overnight. To purify, precipitated into diethyl ether (200 mL) and collected a yellow solid. This solid was twice washed with DI water, then redissolved in ethanol and precipitated again into diethyl ether. The solid was dried to 115 mg, with 1 H NMR confirming absence of remaining small molecule species and UV-Vis spectroscopy (ε = 49,000 cm -1 M -1 in MeOH) determined extent of end-group functionalisation to be approximately 81%. 1a coum . This was prepared using analogous method described for preparation of 1a by replacing 1 with 1 coum . Degree of ionization was determined to be 58% by 1 H NMR (Figure S22), M n,NMR = 19.9 kDa. 1b coum . This was prepared using analogous method described for preparation of 1b by replacing 1 with 1 coum . Degree of ionization was determined to be 55% by 1 H NMR ( Figure S23), M n,NMR = 13.8 kDa. The hazy yellow solution was then deoxygenated by argon bubbling for 5 minutes. Ethylamine (25 μL of 2 M solution in THF, 50 μmol) and P(OEt 3 ) (10 μL, 60 μmol) were then added, resulting in slight loss of yellow colour in solution. After stirring for 2.5 h at RT, Coumarin maleimide (4.5 mg, 12 μmol) was added and the solution was left stirring overnight. To purify, precipitated into diethyl ether (200 mL) and collected a yellow solid. This solid was dissolved in DI water and dialysed against DI water 3 times. Freeze-dried the retentate to 77 mg, with 1 H NMR confirming absence of remaining small molecule species. UV-Vis spectroscopy (ε = 49,000 cm -1 M -1 in MeOH) determined extent of end-group functionalisation to be approximately 53%. 2a coum . This was prepared using analogous method described for preparation of 2a by replacing 2 with 2 coum . Degree of ionization was determined to be 57% by 1 H NMR (Figure S24), M n,NMR = 49.9 kDa. 2b coum . This was prepared using analogous method described for preparation of 2b by replacing 2 with 2 coum . Degree of ionization was determined to be 49% by 1 H NMR ( Figure S25) M n,NMR = 42.0 kDa.
During initial dialysis some blue coloration from the Cy5 dye was observed in the permeate, however an intense blue coloration remained in the retentate after complete dialysis, indicating successful Cy5 functionalisation.

Cell culture (transfection)
For GFP transfection ( Figure S27). Cell suspension measurements (5.0 x 10 3 cells, n = 4) of GSH were converted to approximate intracellular GSH concentration by assuming a cell volume of 1.7 pL. 38

Cell culture (cellular uptake)
pEGFP pDNA was labelled with Cy3 (LabelIT Mirus Bio, pDNA Cy3 ) according to manufacturer's instructions. Cells were seeded in 8-well glass bottomed plates at a density of 1.5 x 10 4 cells per well, with 0.2 mL cell culture medium and incubated overnight. The medium was then replaced by fresh cell culture medium (0.2 mL) and pDNA complex solutions were added (15 μL for polyplexes and micelles, and 30 μL for lipopolyplexes; all prepared using pDNA Cy3 and coumarin labelled polymers: 1a coum , 1b coum , 2a coum , 2b coum ). Each treatment delivered 1.0 μg pDNA / well (5 μg/mL). The cells were then incubated for 4 h, after which the cells were washed three times with cell culture medium. The cells were then imaged with CLSM (Zeiss LSM 710) using a x40 objective (Fluar 40x/1.30 Oil M27, Zeiss) with λ ex = 405 nm, λ det = 410 -538 nm for coumarin and λ ex = 543 nm, λ det = 548 -797 nm for Cy3. Each well was imaged at least 3 times in both bright field and Coumarin/Cy3 fluorescent channels. The images were processed using Fiji (Image J) software, with cells sectioned manually in bright field and the area average fluorescence across all cells evaluated in Coumarin and Cy3 channels.
For uptake of responsive cationic moiety study, cells were seeded in 8-well glass bottomed plates at a density of 1.5 x 10 4 cells per well, with 0.2 mL cell culture medium and incubated overnight.
The medium was then replaced by fresh cell culture medium (0.2 mL) and pDNA complex solutions were added (30 μL, lipopolyplexes prepared using pEGFP and the Cy5 labelled polymer

Cell culture (cytotoxicity)
Cytotoxicity was evaluated using a commercial MTS assay according to manufacturer's instructions (CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay, Promega). Cells were seeded in 96-well plates at a density of 5.0 x 10 3 cells per well, with 0.1 mL cell culture medium and cultured overnight. The medium was then replaced by fresh cell culture medium (0.1 mL) and pDNA complex solutions were added (for polyplexes 7.7 μL and lipopolyplexes 15.4 μL). Each treatment delivered 0.5 μg pDNA / well (5 μg/mL). The cells were then incubated for 48 h, after which 20 μL CellTiter 96 ® AQ ueous One Solution Reagent was added to each well, with the cells incubated for a further 2 h. The absorbance at 490 nm was then measured for each well using a plate reader and each treatment was measured in triplicate. Cell viability was then evaluated by subtracting the absorbance recorded for no cells and dividing this value by that from untreated cells. Figure S1. Example 1 H NMR (D 2 O) spectra from reaction of cationic units on 1a (1a + ) with threonine (Thr) to form the neutralised units on 1a (1a 0 ) and side product DVP-Thr. Spectra split into 3 differently scaled sections for clarity. Integrals were standardised to an ethanol internal standard (*), with cationic conversion evaluated based on red (1a + ), and blue (1a 0 ) signals. See   against DI water (3x 30 mL) (Dialysis 3x). Freeze-dried and observed ionization reduced from 66% to 27%, with a corresponding amount of DVP regenerated (Freeze-drying). Exchanging the acetate counterion with a chloride (dialysis with NaCl) allows for isolation through freeze-drying without loss in ionization.