Gene therapy is broadly defined as the procedure used to genetically modify the target cells with the intention of altering gene expression to prevent, relieve, or reverse pathological genetic deficiency conditions including both inherited and acquired diseases. Over the past two decades, the high efficacies of trans-gene expression by viral vectors has made them attractive and advanced their applications in numerous clinical trials. However, vector-host safety profiles are adversely affected by side effects of viral vectors, including immunogenicity [1
], carcinogenesis [2
] and broad tropism [3
]. The technology of viral gene therapy is also hampered by its limited nucleic acid packaging ability [4
] and difficulties associated with the scalability of virus production [5
]. In the area of non-viral gene delivery systems, the utilization of cationic polymers in clinical applications has increased from 2004 to 2017 while viral products saw a dramatic decrease [6
]. The efforts to develop enhanced polymeric vectors resulted in major advances such as larger gene delivery capacities, lower immunogenicity and easier manufacturing processes compared to the current viral vector technology [7
]. Encouragingly, linear poly(β-amino ester)s (LPAEs), one type of the most versatile polymeric vectors first developed in 2000 [9
], demonstrated its superiority in gene transfection in comparison with other non-viral vectors. After establishing a polymer combinational library, over 2500 LPAEs have been synthesized and screened [10
]. The backbone, side chain, terminal group and degradable linkages in poly(β-amino ester)s (PAEs) can be adjusted easily [12
], the polymer/DNA nanoparticles are biodegradable with a half-life between 1 and 7 h in an aqueous environment [15
], and they are not cytotoxic. These polymeric nanoparticles can be fabricated, lyophilized, frozen and stored for at least two years without a reduction of efficiency [16
], suggesting that PAEs may provide promising alternatives for future translational applications. Among all the LPAEs, the top-performing poly(5-amino-1-pentanol-co
-1,4-butanediol diacrylate) (C32) series have been shown to achieve comparable efficacy to adenovirus in human primary cells [17
] and umbilical vein endothelial cells [18
]. Notably, among the end-modified C32 polymeric vectors, the one end-capped with 1,3-diaminopropane (termed as LC32-103), has been identified to be an extraordinarily efficient vector both in vitro and in vivo in a wide range of biomedical applications [17
Structure is a crucial parameter determining aspects of a gene vector such as efficiency and safety. Branched polymers have many attractive properties over linear polymers including a three-dimensional (3D) structure with multiple functional terminal groups and relatively high molecular flexibility [21
], offering many reaction units for further conjugation. To date, branched polyethyleneimine (PEI) [23
], branched poly-l
-lysine (PLL) [24
], branched poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) [26
] and branched glycopolymers [27
] have been proven to be superior to their linear rivals in transfection efficiency. Although the linear structure of linear C32 (LC32) is limited by only two end groups, branched C32 (HC32) has rarely been synthesized. There are challenges in the synthesis of highly branched poly(β-amino ester)s (HPAEs) due to their very restrictive reaction conditions (e.g., high pressure and presence of a special catalyst [28
], or unequal reactivity of the multiple functional groups in monomers [29
]). Recently, by introducing trimethylolpropane triacrylate (TMPTA), a branching monomer, HPAEs were synthesized from the same library of LPAE monomers by our group. HPAE-mediated transfection efficiency was enhanced up to 8521-fold over the corresponding linear counterparts and the commercially available reagents PEI and SuperFect [30
To our knowledge, there is no study on HC32 with high degree of branching. Therefore, branching of the LC32 needs to be further explored to investigate how the structure affects the transfection efficiency. Herein, based on LC32-103, we successfully developed a series of HC32-103 polymers with diverse degrees of branched structures. Furthermore, physicochemical properties, size, zeta potential of polyplexes, proton buffering capacity, DNA binding affinity, and polyplex stability in the presence of serum and salts were investigated to understand the underlying mechanism behind HC32 in gene delivery. Finally, utilizing Gaussia luciferase (Gluciferase) and green fluorescent protein (GFP) coding plasmid DNA as reporter genes, the performance of gene delivery of HC32 was further compared with LC32-103, placing emphasis on both transfection efficiency and cytotoxicity.
2. Materials and Methods
Chemicals 1,4-butanediol diacrylate (C, VWR, Dublin, Ireland, 98%), 5-amino-1-pentanol (32, Sigma, Dublin, 99%), 1,3-diaminopropane (103, Sigma, Dublin, Ireland, 99%), trimethylolpropane triacrylate (Sigma, Dublin, 99%), lithium bromide (LiBr, Sigma, Dublin, Ireland, 99%), solvents dimethyl sulfoxide (DMSO, Sigma, Dublin, Ireland, 99%), dimethylformamide (DMF, Fisher Scientific, Dublin, Ireland, 99%), diethyl ether (Sigma, Dublin, Ireland, 99%), deuterated chloroform (CDCl3, Sigma, Dublin, Ireland, 99.9%), Hank’s balanced salt solution (HBSS, Sigma, Dublin, Ireland), branched polyethyleneimine (PEI, Mw = 25 kDa, Sigma, Dublin, Ireland), SuperFect (Qiagen, Dublin, Ireland), BioLuxTM Gaussia Luciferase Assay Kit (New England Biolabs, Dublin, Ireland) and Alamarblue Assay Kit (Invitrogen, Dublin, Ireland) were used as received. Sodium acetate (Sigma, Dublin, Ireland, pH 5.2 ± 0.1, 3 M) was diluted to 0.025 M prior to use. Picogreen was purchased from Life Technologies (Dublin, Ireland). Cell culture Dulbecco’s modified Eagle Medium (DMEM) was purchased from Sigma (Dublin, Ireland). Keratinocyte Growth Medium 2 (c-20011) was purchased from PromoCell (Dublin, Ireland). Penicillin-steptomycin was purchased from Thermo Fisher Scientific (Dublin, Ireland). Fetal bovine serum (FBS, Gibco, (Dublin, Ireland) was filtered through 0.2 μm filters before use. Cell-secreted Gaussia princeps luciferase plasmid (pCMV-GLuc) and GFP plasmid (pCMV-GFP) were obtained from New England Biolabs, London, UK.
2.2. Polymer Synthesis
LC32 and HC32 base polymers (polymers prior to end-capping) were synthesized first. Monomer feed ratios for the synthesis of LC32 and HC32 base polymers are listed in Table S1
. Taking HC32-10%-103 synthesis as an example, typically, 1,4-butanediol diacrylate (2 mmol, 0.396 g), trimethylolpropane triacrylate (0.2 mmol, 0.0592 g) and 5-amino-1-pentanol (2 mmol, 0.206 g) are dissolved in 1 mL DMSO, and the reaction occurs at 90 °C. Agilent 1260 Infinite gel permeation chromatography (GPC) was used to monitor the evolution of molecular weight (Mw
). The reaction was stopped by diluting the mixture to 100 mg/mL with DMSO when Mw
was approaching 10,000 Da. 288 μL (2.64 mmol) 1,3-diaminopropane was added to end-cap the acrylate terminated base polymer at room temperature for 48 h. After that, polymers were precipitated into diethyl ether three times and dried under vacuum for 48 h before being stored at −20 °C.
2.3. Molecular Weight Measurements
Molecular weight (Mw), polydispersity index (PDI) and Mark-Houwink alpha parameter (MH Alpha) 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 monitor the Mw of polymers during the polymerization process, 20 μL of the reaction mixture was collected at different time points, diluted with 1 mL of DMF, filtered through a 0.2 μm filter and then 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. Columns were calibrated with linear poly(methyl methacrylate) (PMMA) standards.
2.4. Proton Nuclear Magnetic Resonance (1H NMR)
Chemical structure and composition of polymers were confirmed with 1H NMR. Polymer samples were dissolved in CDCl3. Measurement were carried out on a Varian Inova 400 MHz spectrometer (Edinburgh, UK) and reported in parts per million (ppm) relative to the response of the solvent (7.24 ppm) or to tetramethylsilane (0.00 ppm).
2.5. Acid-Base Titration
To determine the proton buffering capacity, acid-base titration was conducted. 10 mg polymers were dissolved in DMSO to 100 mg/mL stock solution and then diluted with deionized water to 0.2 mg/mL, the pH value was initially adjusted to 10.0 using 1 M NaOH and then titrated to 3.0 with HCl (0.01 M). The pH values were determined with a Sartorius PB-10 pH meter and the increment was 100 μL.
2.6. Picogreen Assays
DNA binding affinity of polymers was measured with Picogreen assays. Briefly, polymers were dissolved in DMSO to 100 mg/mL. According to the w/w ratio, DNA and polymers were diluted with 30 μL of sodium acetate buffer, mixed by vortex for 15 s, and allowed to incubate for another 10 min. Two micrograms of DNA was used for each sample preparation. Then, 60 μL of Picogreen solution, which was prepared according to supplier’s instructions, was added and allowed to incubate for another 5 min. To a 96-well plate, 200 μL of DMEM (without serum) was added, and then 30 μL of the polyplex solution was added. Fluorescence measurements were carried out with a plate reader with an excitation at 490 nm and an emission at 535 nm.
2.7. Size and Zeta Potential of Polyplexes
Typically, 1 μg of DNA was used for each sample. According to the polymer/DNA weight ratio (w/w), DNA and the required polymer were dissolved in 40 μL of sodium acetate, respectively. Then, the polymer solution was added to the DNA solution and mixed by vortex for 15 s. The mixture was kept still for 10 min to formulate polyplexes. After that, 1 mL of deionized water was added and polyplex sizes and zeta potentials were measured with a Malvern Instruments Zetasizer (Nano-2590) (Malvern, UK) at a 90° scattering detector angle. All the measurements were repeated four times.
2.8. Polyplex Stability Measurements
To measure polyplex stability in the presence of serum, polyplexes were prepared as mentioned above and then FBS was added to the polyplex solution; the final FBS concentration was 5% or 10%. Polyplex sizes were measured immediately, as well as 30, 60 and 120 min post-incubation. To measure polyplex stability under high ion concentration, sodium chloride (NaCl) solution was added into the polyplex solution, and the final concentration was 300 or 600 mmol, respectively. Polyplex sizes were measured at 0, 30, 60 and 120 min post-incubation. All the measurements were repeated four times.
2.9. Polymer Degradation
Polymers were first dissolved in DMSO to 100 mg/mL and then diluted with deionized water to 1 mg/mL. The mixtures were incubated at 37 °C under stirring. At 0, 1, 2 and 4 h post-incubation, 10 mL of the polymer solution was taken out and freeze-dried immediately, Mw of the degraded products were measured as mentioned above. The percentage of degradation was defined as the molecular weight of the degraded polymers divided by the Mw of the original polymers.
2.10. DNA Release
To measure the DNA release at different time intervals, polyplexes were prepared as mentioned above in sodium acetate buffer and then diluted with deionized water. Fluorescence was measured with Picogreen assays as mentioned immediately, 1, 2 and 4 h post-incubation.
2.11. Cell Culture
The human cervical cancer cell line HeLa was cultured in DMEM containing 10% FBS and 1% Penicillin/Streptomycin (P/S). RDEBK cells were cultured in keratinocyte growth medium 2 (c-20011 pROMOCELL) with 1% Penicillin-steptomycin. Cells were cultured at 37 °C, in a humid incubator with 5% CO2, under standard cell culture techniques.
2.12. Transfection Experiments
Prior to transfection, cells were seeded on 96-well plates at a density of 1 × 104
cells/well in 100 μL media and cultured until 70–90% confluence. We optimized the commercial transfection reagents PEI and SuperFect according to the manufacturer’s instructions, and also referred to one previous publication [26
]. The commercial reagents were used in optimized formulations: w
= 3:1 (PEI) or w
= 9:1 (SuperFect). A measure of 0.5 μg DNA was used for both cell types. Polyplexes were prepared as mentioned below: Briefly, the polymer and DNA solutions were diluted with sodium acetate buffer to equal volumes (10 μL) according to the w
ratios. Then, polymer solutions were added to the DNA solution, vortexed for 10 s and allowed to stand for 10 min. Cell culture media was then added to increase the volume of the polyplex solution to 100 μL. The media in the wells of the cell culture plates was removed quickly and the polyplex solution was added. Four hours later, the transfection medium was replaced with fresh medium and the cells were cultured for another 44 h.
2.13. Evaluation of Gene Transfection Efficiency Using Gluciferase Assays and GFP Expression
At 48 h post-transfection, using Gluciferase DNA, measurements of the Gluciferase activity were carried out as per the provided protocol using a SpectraMax M3 plate reader (Dublin, Ireland) with Gluciferase activity directly detected in the cell supernatant and plotted in terms of relative light units (RLU). Measurements were performed in quadruplicates with error bars indicating ± standard deviation. The GFP expression was observed and imaged by an inverted fluorescence microscope (Olympus IX81, Dublin, Ireland).
2.14. Cytotoxicity Assessment after Transfection Using Alamarblue Assay
To perform Alamarblue assay, cell supernatants were first removed and then cells were washed with HBSS, followed by the addition of 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. Measurements were performed in quadruplicates with error bars indicating ± standard deviation.
All quantitative data were analyzed using GraphPad Prism (v.5, GraphPad Sofeware, San Diego, CA, USA). D’Agostino and Pearson omnibus normality tests were used to determine normal distribution. A one-way ANOVA was used where normal distribution was evident, followed by Tukey’s post hoc test. p values < 0.05 was considered to be statistically significant. All quantitative data were expressed as mean ± standard deviation.