Highly branched poly(β-amino ester) delivery of minicircle DNA for transfection of neurodegenerative disease related cells

Current therapies for most neurodegenerative disorders are only symptomatic in nature and do not change the course of the disease. Gene therapy plays an important role in disease modifying therapeutic strategies. Herein, we have designed and optimized a series of highly branched poly(β-amino ester)s (HPAEs) containing biodegradable disulfide units in the HPAE backbone (HPAESS) and guanidine moieties (HPAESG) at the extremities. The optimized polymers are used to deliver minicircle DNA to multipotent adipose derived stem cells (ADSCs) and astrocytes, and high transfection efficiency is achieved (77% in human ADSCs and 52% in primary astrocytes) whilst preserving over 90% cell viability. Furthermore, the top-performing candidate mediates high levels of nerve growth factor (NGF) secretion from astrocytes, causing neurite outgrowth from a model neuron cell line. This synergistic gene delivery system provides a viable method for highly efficient non-viral transfection of ADSCs and astrocytes.

protocols. Green fluorescent protein plasmid (pCMV-GFP) was purchased from Aldevron. Cell secreted Gaussia princeps luciferase plasmid (pCMV-Gluc) were obtained from New England Biolabs. NGF plasmid pEX-A2-NGF (2.4 Kbp) was constructed by Eurofins. BioLux TM Gaussia luciferase assay kits, enzymes XbaI and BamHI obtained from New England Biolabs and alamarBlue ® from Invitrogen were used as per protocols. Human beta-NGF protein were purchased from Sino Biological. DNA ladders was purchased from Thermo Fisher Scientific. Human beta-NGF ELISA Kits were purchased from Sigma and used as per the manufacturer's protocol.

Instrumentation
Chemical structure and composition of polymers were verified with 1 H NMR spectra, which was

Polyplex preparation
HPAE/DNA polyplexes were prepared according to previous published procedures with slight modification [3][4][5][6] . HPAEs were first dissolved in DMSO (for molecular biology) by vortexing to give 100 mg/mL DMSO stock solutions and kept at -20 °C for the following studies. DNA was diluted with TE buffer to 1 mg/mL and kept at 4 °C for the following studies. The HPAE/DNA weight ratio (w/w) used in this work was 10:1 and 20:1. Taking the use of 1 μg DNA for polyplex preparation as an example: 1 μL DNA stock solution was added to 19 μL sodium acetate (pH 5.2, 0.025 M) and mixed by vortexing for 3-5 seconds. According to the w/w ratio of 10:1 or 20:1, 0.1 or 0.2 μL DMSO stock solution was added to 19.9 or 19.8 μL sodium acetate (pH 5.2, 0.025 M) and mixed by vortexing for 10-15 seconds to make sure the HPAE was dissolved completely in sodium acetate.
Then, the HPAE solution was added into the DNA solution and mixed by vortexing for 10-15 seconds. The mixed solution was kept undisturbed for 10-15 minutes to allow HPAE/DNA polyplex formation. After that, dependent on the specific application, the polyplex solution was diluted with different media. When other amounts of DNA were used for polyplex preparation, the volume of the HPAE DMSO stock solution and sodium acetate buffer was adjusted accordingly.

DNA binding affinity of HPAEs
DNA binding affinity of HPAEs was quantified utilizing Picogreen assays. Briefly, polyplexes were prepared as above and 0.5 μg DNA was used for each sample. Then picogreen solution was added into the polyplex solution, the mixture was incubated for 5 minutes followed by the addition of 200 μL DMEM media. The mixture was then transferred to a black 96-well plate. Fluorescence of the solution was measured at a 490 nm excitation wavelength and a 535 nm emission wavelength. 5 The mixture of DMEM, DNA and Picogreen was used as control.

Transmission electronic microscopy (TEM) characterization of polyplex morphologies
To characterize polyplex morphologies, LPAE, HPAESS-3, HPAESS-4 or HPAESG-1 and DNA were diluted in sodium acetate and then mixed in equal volumes (1 μg DNA, w/w = 20:1). After 15 min incubation, the polyplexes were centrifuged at high speed for 1 min and then washed twice with deionized water. Afterwards, the polyplexes were re-suspended in water, 5 μL of polyplex solution was dropped on a holey carbon film on 200 mesh copper grids and freeze dried for 1 hour. Samples were observed with TEM (FEI Tecnai 120) with an accelerating voltage of 120 kV.

MC GFP preparation
The production of MC GFP was carried out according to our previous report 7

Cytotoxicity of the degraded byproducts of HPAE-3 and LPAE
Cytotoxicity of the degraded byproducts of HPAE-3 and LPAE were tested using HKC8 cells. Cells were seeded in 96-well plates at a density of 3 × 10 4 cells per well in 100 μL of cell culture media 6 and cultured until 80-90% confluence. The degraded byproducts of HPAE-3 and LPAE were dissolved in DMSO to 500 mg/mL. And then, according to the final concentration, the required amount of byproducts-containing DMSO solution was added into the cell culture mediate directly.
After another 24 hours of incubation, the cell viability was measured using the alamarBlue reduction assay as per manufacturer's protocol. Briefly, cell culture media was removed, cells were washed with HBSS twice, and then 100 µL 10% alamarBlue HBSS solution was added and the cells were incubated for another 2 hours. Next, the alamarBlue solution was transferred to a black flat bottomed 96-well plate. Fluorescence was measured using the standard protocol. Cells without any treatment were set as 100% viable.

Evaluation of the gene transfection activity of HPAEs, HPAESSs and HPAESGs
One day prior to transfection, cells were seeded in 96-well plates at a density of 1~3 × 10 4 cells per well in 100 μL of cell culture media and cultured until 70-80% confluence. rADSC and hADSC used for transfection were under passage six, Neu 7 and primary astrocytes used for transfection were under passage five. 0.5 μg DNA per well was applied in HKC8 and HeLa, while 0.25 μg DNA per well was applied in rADSC, hADSC, Neu7 and primary astrocytes. To prepare polyplexes, HPAEs were dissolved in DMSO to 100 mg/mL as stock solutions, and then according to polymer to DNA weight ratios (w/w), varying stock solutions were diluted to 10 µL sodium acetate buffer.

Polyplex endo/lysosomal escape study
Cells were seeded in 96 well plate at a density of 3 × 10 3 cells per well in 100 μL media. Using Cy3 labelled DNA, polyplxes were prepared and the gene transfection was carried out at w/w = 20:1. 4 hours later, the acidic endo/lysosomal vesicles of cells were stained with Lyso-Tracker Green at 37 o C for 1 minute. Cells were then imaged immediately using a fluorescent microscopy. Pearson Correlation Coefficient (PCC) of the cells after transfection with HPAE-3/DNA and LPAE/DNA polyplexes were calculated using ImageJ from three images.

Gene transfection HPAESG/MC DNA in ADSCs and astrocytes
To evaluate the effectiveness of the optimized HPAESG in combination with MCDNA in gene transfection, cells were seeded in 24-well plates at a density of 5 × 10 4 cells/well in 500 μL media and cultured until 70-80% confluence. Transfection was performed as mentioned above with 1 μg MC DNA per well. To demonstrate the superiority of MC DNA, a normally sized DNA (5.6 kbp) was also used to carry out all the transfection in the same manner for comparison. 48 hours post transfection, the GFP positive cells were visualized and imaged with a fluorescent microscope. The transfection efficiency was analyzed via flow cytometry. Cells were collected as per standard protocols, at least 8,000 cells were counted for each sample. Cells that received only DNA were used as controls.

HPAESG-1/MC NGF transfect astrocytes
The effectiveness of HPAESG-1 in combination with MC NGF to mediate the expression and 8 production of NGF in Neu7 and primary astrocytes was studied. The gene transfection parameters including cell density (5 × 10 4 or 2 × 10 5 cells/well, 24 well plates), DNA dosage (1 or 2 μg/well), with or without serum and transfection time (24, 48 or 72 hours) were optimized systematically.
Briefly, cells were seeded in 24-well plates and cultured until 80-90% confluence. Transfection in the presence of serum was performed as mentioned above. For serum free transfection, cells were first incubated with HPAESG-1/MC NGF polyplexes in the absence of serum for 4 hours, and then the cell culture media was replaced with 10% FBS containing media. PP NGF where the MC NGF was derived from was used as a control for comparison, and all the transfection was carried out in exactly the same manner. There is a GFP tag with the MC NGF and PP NGF, therefore, after 24, 48 and 72 hours, the GFP expression was observed with a fluorescence microscope and the transfection efficiency was quantified with flow cytometry. Cell viability was analyzed with alamarBlue assays. Meanwhile, the cell supernatants were collected and the NGF concentration was measured with enzyme-linked immunosorbent assay (ELISA) as per the manufacturer's protocol.

Bioactivity of NGF produced by astrocytes after transfection.
PC12 cells were seeded in 96-well plates at a density of 1 × 10 3 cells/well in 100 μL media and incubated overnight to attach. And then, the media on the cells was replaced with standard PC12 cell culture media (DMEM with a high glucose concentration, PC12 media group), standard Neu7 cell culture media (DMEM with a low glucose concentration, Neu7 media group), conditioned Neu7 cell culture media (media saved from Neu7 cells after transfection with HPAESG-1/MC NFG, the concentration of NGF was determined to be 26.9 ng/mL, Conditioned Neu7 media group) and standard PC12 cell culture media but with 50 ng/mL recombinant NGF (PC12+NGF group), respectively. Over the following two weeks, the media on the cells were changed with the corresponding media as mentioned above every 3 days. The outgrowth of neurites was checked with a fluorescent microscope daily. At day 7 and 14, the cells were washed with PBS slightly three times and then fixed with paraformaldehyde (PFA) following the standard fixation procedures. The cell nucleus and F-actin were stained with DAPI (blue) and Alexa Fluor 594 (red), as per the manufacturers' protocols, respectively. And then, the cells were observed with a fluorescent microscope to check the neurite outgrowth.

Direct transfection of PC12 with HPAESG-1/MC NGF
PC12 cells were seeded in 24-well plates at a density of 5 × 10 3 cells/well in 500 μL media. The next day, with 1 μg DNA per well, the cells were transfected with HPAESG-1/MC NGF or HPAESG-9 1/PP NGF in the absence of serum as mentioned above. The cell culture media was changed every 3 days. One week post transfection, GFP expression and neurite outgrowth were checked with a fluorescent microscope, the efficiency of GFP and NGF expression was measured with flow cytometry and ELISA, respectively.

Statistical analysis
All transfection data were analyzed using GraphPad Prism version 8 (GraphPad Software). A oneway ANOVA was performed, followed by Dunnett method as the post hoc test. P values < 0.05 were considered to be statistically significant. Additionally, data of the GFP positive cell percentage was analyzed using paired Student's t-test. All transfection experiments were performed in quadruplicate unless otherwise stated, with error bars indicating ±SD.