Mitigated Cytotoxicity and Tremendously Enhanced Gene Transfection Efficiency of PEI through Facile One‐Step Carbamate Modification
Authors: Chuan Yang, Wei Cheng, Pei Yun Teo, Amanda C. Engler, Daniel J. Coady, James L. Hedrick, Yi Yan Yang
Journal: Advanced Healthcare Materials (2013)

Extremely efficacious gene transfection vector: The rapid and facile modification of PEI with commercially available TMC produces an extremely efficacious gene delivery vector with minimal cytotoxicity. Functionalization of PEI is easily controlled by PEI:cyclic carbonate feed ratios and allows for the addition of functionality. Modified PEIs hold great potential as gene delivery systems due to easy synthesis, scalability, low cost, low toxicity, and outstanding transfection capacity Gene therapy has been heralded as a potential solution to numerous genetic diseases such as haemophilia,1 cystic fibrosis2 and even cancer.3 Therapeutic action is achieved via knocking down errant genes or expressing therapeutic genes through gene delivery, which to date remains a limiting challenge.4 Gene delivery vectors are generally divided into two major categories: viral and non-viral. Viral vectors are extremely effective at delivering genes, however, they are limited by relatively low plasmid loading capacity, immunogenicity, and safety issues that hinder their clinical applications.5 Alternatively, non-viral gene delivery vectors are receiving a tremendous amount of interest because they are relatively inexpensive, reproducible and synthetically modular.6-11 Among non-viral carriers, cationic polymeric systems have been extensively investigated. In particular, branched polyethylenimine (PEI, Mn 10 kDa) has been shown to be one of the most efficient synthetic gene delivery carriers in vitro,6, 12 and has been used as the gold standard for in vitro gene transfection. The success of PEI for gene transfection is primarily attributed to its high density and composition of primary, secondary and tertiary amines. Primary amines are protonated at physiological pH 7.4 and enable PEI to bind negatively charged gene effectively into nano-sized complexes via electrostatic interactions. The secondary and tertiary amines are able to sequester protons in the endosomes. This buffering effect leads to the influx of chloride ions, which causes osmotic swelling and finally the rupture of the endosomes–a phenomenon known as the "proton-sponge" hypothesis.6, 13 However, despite PEI's superficial successes, its use in vivo is limited by its high toxicity brought about by its high cationic charge density.14, 15 Cytotoxicity reduction has been attempted through modifying primary amines on PEI. For example, Wang et al. recently incorporated cystamine derivatives containing a reductively cleavable disulphide bond, via the primary amines of PEI to facilitate intracellular release of bound DNA and reduce charge density of the polymer in acidified endosomes.16 This method demonstrated reduced cytotoxicity and almost a two-fold enhancement of luciferase expression efficiency in 293T cells when compared to unmodified PEI. Additionally, the introduction of a methyl group to cystamine further improved transfection efficiencies. Thomas et al. showed that hydrophobic modifications of PEI by acylation with alanine resulted in a 2-fold increase in luciferase expression efficiency and reduced cytotoxicity for COS-7 cells.17 Moreover, the acetylation of PEI18 and modification with N- isopropylacrylamide19 were also found to reduce cytotoxicity and increase gene expression efficiency. The incorporation of hydrophobic groups has been suggested to increase polymer/DNA complex interactions with the cell membrane and therefore enhance endocytosis of the complexes, which in turn leads to enhanced transfection efficiencies.17 Although improved gene transfection and reduced cytotoxicity have been achieved through blocking primary amines on PEI, these modifications are often laborious or synthetically challenging. In this study, cyclic carbonates were used to modify primary amines on branched PEI (Mn 10 kDa) through a single step, spontaneous ring-opening event (Figure 1a). Commercially available trimethylene carbonate (TMC) at various PEI:TMC ratios (1, 8, 25, and 100) was substituted onto PEI to determine the optimum substitution ratio for gene delivery carriers with high transfection efficiency and minimal cytotoxicity. This optimum PEI:cyclic carbonate ratio was then used to explore the impact of different substituted cyclic carbonates on transfection efficiency and cytotoxicity. Single-step carbamate modifications of branched PEI (a, PEI-TMC, PEI-ethyl, PEI-benzyl and PEI-urea), typical 1H NMR spectrum (b, PEI-TMC(25)), and particle size and zeta potential of PEI-MTC(25)/DNA complexes (c). Modified PEI condensed DNA into nanosized particles with reduced zeta potential as compared to unmodified PEI, which are desirable for gene transfection. Reaction of nucleophilic PEI with cyclic carbonates was done only for one hour under ambient conditions without using a catalyst or producing any byproducts. The ring-opening event converts primary amines into carbamate linkages and primary hydroxyl groups, while concurrently installing functionality present on the original cyclic carbonate20, 21 (Figure 1a). The final polymer composition, or degree to which PEI was functionalized, was readily controlled by PEI:cyclic carbonate feed ratios (Table S1 in Supplementary Information). Modification was monitored and quantified using 1H NMR. For example, TMC incorporation was determined by comparing relative integral intensities of PEI methylene signals (2.4-2.9 ppm) and TMC methylene signals (1.83 ppm) (Figure 1b). The obtained values were experimentally identical to the starting PEI:TMC feed ratio, hence suggesting quantitative reaction (Table S1). This clean and controlled substitution reaction permitted a correlation study between PEI polymer functionalization and resulting transfection ability. It should be noted that when excess TMC relative to the PEI primary amines was used (PEI contains 58 primary, 116 secondary and 58 tertiary amines),22 the secondary amines were also found to react with the cyclic carbonates, as in the case of modification with 100 TMC groups (Table S1, TMC 1:100). The condensation of DNA by the polymers to form complexes of relevant size and surface charge is an important requirement for gene delivery. The gene binding efficiency of modified PEIs was studied by measuring the fluorescence emitted by SYBR green dye when bound to free DNA.23 For most polymers, the relative fluorescence decreased with increasing N/P ratio (molar ratio of nitrogen content in PEI to phosphorus content of DNA), indicating the inaccessibility of the dye to the condensed DNA (Figure S1). Modification of PEI with an increasing number of TMC up to 25 slightly increased the IC50 value (N/P ratio at which the relative fluorescence is 50%) of the polymers as compared to unmodified PEI (IC50 value: ∼1.8 for PEI, ∼2.1 for PEI-TMC(1) and PEI-TMC(8), and ∼2.5 for PEI-TMC(25)) (Figure S1), indicating marginally reduced gene binding ability. In contrast, PEI-TMC(100) showed a drastic reduction in gene binding capacity with an IC50 value of more than 10. This drastic reduction is most likely a result of complete blockage of primary amines, which are required for gene binding. Modification of PEI with PEI:cyclic carbonate ratio of 1:25 for the other hydrophobic groups (ethyl, benzyl and urea) (Figure S2) also increased the IC50 values to some extent (∼2.8 vs. ∼1.8). Particle size and zeta potential are also important parameters for dictating cellular uptake by non-specific endocytosis.24 All modified PEIs, especially at PEI:cyclic carbonate ratio of 1:25, were found to form DNA complexes ∼100-110 nm in diameter. The zeta potentials for the nanoparticles were found to be ∼12-15 mV (Figure S3). Both values are generally considered ideal for cellular uptake and gene transfection. Unfortunately, positively charged particles suffer from plasma protein and erythrocyte adhesion, which might activate the alternative complement pathway, limiting in vivo applications.25 Therefore, lower zeta potentials of modified PEI/DNA complexes at N/P 20-50 are expected to exert reduced cytotoxicity as compared to unmodified PEI. Cytotoxicity of unmodified and modified PEIs was evaluated in HepG2 liver carcinoma cells. Comparison of unmodified PEI with carbonate-modified PEIs exhibited large cytotoxicity differences (Figure 2). Moreover, even modification with only a single TMC equivalently rendered PEI-TMC minimally toxic at N/P 50. This cytotoxicity reduction could be due to the formation of a hydroxyl shell after polymer-DNA complexation, which shielded the cationic charge of the polymer/DNA nanocomplexes (Figure S3), rendering them less cytotoxic. Further increasing the TMC modification allowed additional toxicity reduction, albeit relatively mild. Modification using functional carbonates also demonstrated analogous toxicity improvements. Among all systems with 25 carbonate groups, PEI-TMC(25) amd PEI-ethyl(25) demonstrated the greatest reduction of cytotoxicity at N/P 30-50. Although the exact mechanism by which cytotoxicity is reduced remains unclear, it can be reasoned that a decreased primary amine concentration diminishes the charge density of PEI/DNA nanocomplexes (Figure S3), thereby lessening cell membrane rupture and consequently reducing cytotoxicity.26 Viability of HepG2 cells after incubation with unmodified or modified PEI/DNA complexes formed at various N/P ratios. Carbamate modifications reduced cytotoxicity significantly. Transfection efficiency of the modified polymers was investigated and compared to unmodified PEI by GFP transfection studies. GFP transfection efficiency reflects the percentage of transfected cells. For most gene therapy cases, it is important for gene carriers to transfect a high percentage of cells such that a large number of cells incorporate the therapeutic gene. Modification with TMC was first conducted to determine optimal carbamate functionalization for gene transfection (Figure 3a). It was found that 25% functionalization provided the best transfection efficiency (64% at N/P 50) while also maintaining minimal cytotoxicity. This transfection efficiency was 5.5-fold higher than that of the unmodified PEI at N/P 20. In addition, PEI-TMC(25)/DNA complexes at N/P 50 were relatively non-cytotoxic with cell viability of more than 85%, while PEI/DNA complexes at N/P 20 were significantly cytotoxic with cell viability of only ∼50% (Figure 2). The optimized PEI:cyclic carbonate ratio was then applied to the modification of PEI with various substituted carbonates (Figure 3b). Despite additional hydrophobicity (PEI-ethyl, PEI-benzyl, PEI-urea) and hydrogen bonding capacity provided by urea groups27, 28 (PEI-urea), PEI functionalized with commercial available TMC proved to be the most effective gene delivery vector. However, even though all modified PEIs were found to have improved cell viability, only PEI-TMC(8), PEI-TMC(25) and PEI-ethyl(25) had much higher transfection efficiency (regardless of N/P ratio) when compared with unmodified PEI. Benzyl and phenyl urea modifications only provided a slight improvement in gene transfection efficiency at N/P 40-50 as compared to unmodified PEI at N/P 20 possibly because their hydrophobicity was too high.17 A similar phenomenon was also reported by Jia et al that benzyloxyl trimethylene carbonate modification (conducted at 70 °C for 48h) did not significantly improve gene transfection efficiency in HepG2 cells.29 Confocal images in Figure 3c further show that TMC modification yielded a larger number of cells transfected with green fluorescent GFP gene. GFP transfection efficiency (a, b) and confocal images (c) of HepG2 cells after incubation with unmodified or modified PEI/DNA complexes formed at various N/P ratios. In c, N/P 15 was used to form PEI/DNA complexes as they were highly toxic at N/P 20-50. Carbamate modifications, especially TMC and MTC-ethyl at PEI:TMC or MTC-ethyl molar ratio of 1:25, increased gene transfection efficiency tremendously. The intracellular trafficking of the polymer/DNA complexes was monitored by confocal microscopy over a 24h time. Prior to complex formation, the DNA was tagged with YOYO-1, a fluorescent intercalator. This permitted real-time visualization of DNA localization within HepG2 cells. As shown in Figure 4, after 2 h of incubation, DNA (green) delivered by both PEI and PEI-TMC(25) was observed to have entered the cell but remained in the cytoplasmic region. At 6 h, it was clearly seen in both the cytoplasmic region and the nuclei (cyan regions in blue-coloured Hoechst-stained nuclei) of the cells incubated with PEI-TMC(25) as compared to those incubated with PEI. This was also observed at 24 h where almost 50% of the cells treated with PEI-TMC(25)/DNA complexes showed DNA localization in the nuclei, whereas most of DNA molecules were still trapped in the perinuclear region of the cells treated with PEI/DNA complexes. This result suggests that in contrast to unmodified PEI, PEI-TMC(25) provided higher nuclear localization and better cell trafficking kinetics. It is postulated that weaker DNA binding of PEI-TMC(25) (Figure S1) leads to easier intracellular release of DNA and increased vector hydrophobicity causes more favourable plasma membrane and nuclear envelope interactions, hence enhancing gene tranfection efficiency (Figure 3a). Cellular trafficking of DNA. HepG2 cells were treated with unmodified PEI or modified PEI/DNA complexes formed at N/P 50. Blue: Hoechst-stained nuclei; Green: DNA labeled with YOYO-1 dye. PEI-TMC(25) provided higher nuclear localization and faster cell trafficking kinetics of DNA as compared to unmodified PEI. In conclusion, the rapid and synthetically facile modification of PEI with commercially available TMC produced an extremely efficacious gene delivery vector with minimal cytotoxicity. Functionalization of PEI was easily controlled by predetermined PEI:cyclic carbonate feed ratios and permitted a simple means for the addition of functionality. This approach can also be extended to include a plethora of other functional groups such as a targeting signal that recognizes a specific cell type and PEG for improving serum stability of polymer/DNA complexes, and responsive linkages for triggered release of other biologically relevant materials. Modified PEIs are envisioned to hold great potential as gene delivery systems due to easy synthesis, scalability, low cost, low toxicity and outstanding transfection capacity. Supporting Information is available from the Wiley Online Library or from the author. This work was supported by the Institute of Bioengineering and Nanotechnology Biomedical Research Council, Agency for Science, Technology and Research), Singapore, and IBM Almaden Research Center, USA. 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