Branch Unit Distribution Matters for Gene Delivery

As a key nonviral gene therapy vector, poly(β-amino ester) (PAE) has demonstrated great potential for clinical application after two decades of development. However, even after extensive efforts in structural optimizations, including screening chemical composition, molecular weight (MW), terminal groups, and topology, their DNA delivery efficiency still lags behind that of viral vectors. To break through this bottleneck, in this work, a thorough investigation of highly branched PAEs (HPAEs) was conducted to correlate their fundamental internal structure with their gene transfection performance. We show that an essential structural factor, branch unit distribution (BUD), plays an important role for HPAE transfection capability and that HPAEs with a more uniform distribution of branch units display better transfection efficacy. By optimizing BUD, a high-efficiency HPAE that surpasses well-known commercial reagents (e.g., Lipofectamine 3000 (Lipo3000), jetPEI, and Xfect) can be generated. This work opens an avenue for the structural control and molecular design of high-performance PAE gene delivery vectors.

Hank's balanced salt solution and alamarBlue Assay Kit were purchased from Sigma and Invitrogen. Sodium acetate (Sigma) was diluted to 0.025 M before use. PicoGreen was purchased from Life Technologies. Cell culture Dulbecco's modified Eagle Medium (DMEM) was purchased from Sigma. Fetal bovine serum (FBS, Gibco, was filtered through 0.2 µm filters before use. The gWiz-GFP commercial plasmid was obtained from Aldevron, Fargo, ND, USA. Xfect was purchased from Medical Supply Co. Ltd. Lipofectamine 3000 (Lipo3000) transfection reagent was purchased from Biosci. JetPEI was purchased from Polyplus Transfection, Illkirch-Graffenstaden, Strasbourg, France.
Monomer feed ratios and reaction conditions for the synthesis of HPAE base polymers are listed in Table 1. Taking the synthesis of HPAE-A1 as an example, BDA (3.96 g), S5 (2.06 g), and PTTA (0.70 g) were dissolved in DMSO (1.53 mL). Then, the solution was bubbled under argon for 15 mins to remove oxygen. After that, the reaction mixture was merged into the preheated oil bath and react with stirring at 90 o C for the desired time. Agilent 1260 Infinite gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) were used to monitor the reaction. The reaction was stopped by diluting the mixture to 100 mg/mL with DMSO when M w was approaching 10 kDa. E7 (2.51 g) was then added to end-cap the acrylate-terminated base polymer at room temperature for 48 h. After that, HPAE polymers were precipitated into diethyl ether three times for purification and dried under vacuum before being stored at −20 o C.

Elution fractionation
After generating polymers HPAE-A1, HPAE-B1, and HPAE-C1, they were franctionated to obtain different components with a range of molecular weights (HPAE-AS1 to AS5, HPAE-BS1 to BS5, and HPAE-CS1 to CS5). Taking the HPAE-A1 as an example, the fractionation procedure is as follows: HPAE-A1 was dissolved in acetone to a concentration of 100 mg/mL, then the solution was slowly added into the mixed solvent of acetone and diethyl ether (v/v=1/9) under gentle agitation at room temperature. The solvent in the supernatant solution was removed by rotary evaporator and the product was collected as component HPAE-AS1. Then the precipitate was redissolved in acetone and precipitated into another mixed solvent with higher acetone ratio (acetone/diethyl ether = 2/8) to generate component HPAE-AS2. By repeating the stepwise precipitation process, Components HPAE-AS1 to AS5 were obtained. HPAE-BS1 to BS5 and HPAE-CS1 to CS5 were obtained following the same procedure.

Molecular weight and dispersity measurements
Number average molecular weight (M n ), weight average molecular weight (M w ), and dispersity (Ɖ) of polymers were determined by GPC equipped with a refractive index detector (RI), a viscometer detector (VS DP) and a dual angle light scattering detector (LS 15° and LS 90°). To sample the molecular weight of polymers during the polymerization process, 20 µL of the reaction mixture was collected at different time points, and diluted with 1 mL of DMF, then filtered through a 0.2 µm filter, and finally measured by GPC. The columns (PolarGel-M, Edinburgh, UK, 7.5 mm × 300 mm, two in series) were eluted with DMF and 0.1% LiBr at a flow rate of 1 mL/min at 60 °C.

Nuclear magnetic resonance (NMR)
The chemical structure and composition of polymers were confirmed with 1 H NMR.
Polymer samples were dissolved in CDCl 3 . Measurements were carried out on a Varian Inova 400 MHz spectrometer.

Polyplex preparation
Generally, the polymers were initially dissolved in DMSO to stock solutions (100 mg/mL), and the stock solutions were further diluted with 25 mM sodium acetate buffer according to the w/w ratio. DNA was diluted to 0.1 mg/mL with sodium acetate buffer.
The polymer solutions were added into the DNA solution, vortexed for 10 s, and allowed to stand for 15 min.

PicoGreen assays
The polyplex was prepared as described above. 2 µg of DNA was used for each sample preparation. 60 µL of PicoGreen solution was prepared according to the supplier's instructions, added to the DNA and allowed to incubate for another 5 min. Ina 96-well plate, 200 µL of medium (without serum) or water was added, and 30 µL of the polyplex solution was then added. Fluorescence measurements were carried out with a platereader with an excitation at 490 nm and emission at 535 nm. All the measurements were repeated in triplicate.

Size measurement of polyplexes
The polyplex was prepared as described above and their sizes were measured with a Malvern Panalytical Zetasizer (ZTS1240). All the measurements were repeated in triplicate.

Morphology measurement of polyplexes
Morphology of polyplexes was characterized by transmission electron microscopy (TEM). After preparing polyplexes as described above, the polyplex solution was dropped onto 200 mesh copper grids and allowed to dry for 20 mins. The polyplex was further washed with distilled water twice to remove excess salts before imaging. Images were captured on a FEI Tecnai 120 TEM at 120 kV in UCD Conway Imaging Core Center.

Cell culture
RDEBK cells were cultured using standard cell culture techniques in keratinocyte growth complete FAD medium (KCa). HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal bovine serum and 1% penicillin-s. Cells were cultured at 37 o C with 5% CO 2 in a humidified incubator under standard cell culture techniques.

Flow Cytometric Analysis
After 48 h post-transfection, cells were harvested with trypsin-EDTA and washed with PBS, then re-suspended in PBS with 2% FBS. The flow cytometry measurements were carried out on an Accuri C6 system in triplicate. Data analysis was performed using the Cytexpert software.

Polyplex cellular uptake
GFP DNA was labelled with a Cy3 (a red fluorescent dye) labelling kit as per the recommended protocol. HEK cells were seeded in 96-well plates. Gene transfection was conducted as above with 0.5 µg of DNA per well. After 4 hours, the medium was removed, and cells were fixed with 4% paraformaldehyde after washing with PBS three times. Next, the cells were permeabilized with 0.1% Triton X-100 and stained with DAPI, followed by visualization under a fluorescence microscope (Olympus IX81). All the measurements were repeated in triplicate.

Cytotoxicity assessment (alamarBlue assay)
To perform the alamarBlue assay, cell supernatants were first removed, then cells were washed with HBSS, followed by adding 10% alamarBlue reagent in HBSS. Living, proliferating cells maintain a reducing environment within the cytosol of the cell, converting the non-fluorescent ingredient resazurin in alamarBlue to the highly fluorescent compound resorufin. This reduction results in a color change from blue to light red and allows for the quantitative measurement of cell viability based on the increase in overall fluorescence and color of the media. The alamarBlue solution from each well was transferred to a fresh flat-bottomed 96-well plate for fluorescence measurements at 590 nm. Control cells without any treatment were used to normalize the fluorescence values and plotted as 100% viable. All the measurements were repeated in triplicate.