Abstract
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene therapy utilizes gene delivery to deliver genetic material with the goal of treating a disease or condition in the cell. Actual viral vectors may have side effects, while actual systems using metal nanoparticles for gene delivery are toxic. Therefore, we designed here a biocompatible tri-block copolymer PEO20–PPO69–PEO20 as a gene delivery vector [PEO: poly(ethylene oxide); PPO: poly(propylene oxide)]. We studied the conjugation of PEO20–PPO69–PEO20 and DNA using various techniques. Results of gel retardation assay along with zeta potential and dynamic light scattering provide evidence of DNA sequestration. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy show that the PO43− groups of plasmid DNA are primarily involved during nanoconjugate construction. The integrity and functionality of plasmid DNA within the cellular environment is further demonstrated by the expression of green fluorescent protein gene in Escherichia coli. Overall, our findings support the use of block copolymers as delivery systems for mammalian and plant cells.
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Introduction
Viral vectors are favored for nucleic acid transport because of small size, protective core, the presence of receptor-specific moieties and membrane fusogenic elements. However, they have potential side effects that limit their applicability as vectors (Check 2002). Conversely, inorganic nanoparticles provide an opportunity for drug, protein and DNA delivery because of their unique properties. However, environmental toxicity, non-degradability and strong association with different cellular components restrict the use of metal nanoparticles (Daima and Navya 2016; Ghosh et al. 2008; Sokolova and Epple 2008). Therefore, organic materials offer better prospects for delivery applications, since enzymes can easily break them down, reducing the likelihood of hostile environmental impact. Moreover, since cellular environment is composed of organic molecules, it is suitable to employ similar kind of materials for payload delivery applications (Duncan et al. 2006; Satchi-Fainaro and Duncan 2006; Hamley 2003; Kwak and Herrmann 2010). However, organic nanostructures have not been fully explored for gene delivery applications, and it is important to develop new efficient organic materials in order to realize their full potential and to overcome the safety issues.
In this spirit, polyplexes have been designed through entropically driven interactions of DNA and synthetic polymers. Polyplexes can be formed through electrostatic interactions between negatively charged DNA and positively charged groups of polymers (Boussif et al. 1995; Gebhart and Kabanov 2003; Pack et al. 2005). However, the use of cationic polymers in such polyplexes has been noted to strongly condense DNA, which reduces the payload delivery efficiency along with enhanced cytotoxicity (Wong et al. 2007). Therefore, amphiphilic block copolymers have also been screened to enhance DNA vaccination for therapeutic needs as biologically active block copolymers can self-assemble into micellar structures and have spontaneous associations with DNA (Gebhart and Kabanov 2003; McIlroy et al. 2009; Torchilin 2007).
In the present study, a biocompatible tri-block copolymer of poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) [PEO20–PPO69–PEO20] is chosen as a plasmid DNA delivery vector. To understand the interactions between plasmid DNA and PEO20–PPO69–PEO20, a number of sensitive techniques were utilized to establish that PEO20–PPO69–PEO20 is proficient in constructing compact nanoconjugates with plasmid DNA.
Experimental
Plasmid DNA isolation, purification and construction of nanoconjugates
Plasmid DNA containing green fluorescent protein (GFP) and ampicillin resistance genes was isolated and purified according to Shambrook and Russell with some modifications (Sambrook and Russell 2000). Under vigorous stirring, in 100 mL deionized MilliQ water at 50 °C, 10 g of PEO20–PPO69–PEO20 was dissolved to obtain a stock solution. Different volumes of this solution were incubated with a fixed amount of 10 µg plasmid DNA for 2 h at 37 °C to form nanoconjugates.
Preparation of competent cells
Modified CaCl2.MgCl2 method in the presence of tetracycline was employed to make competent cells of Escherichia coli DH5α (Sambrook and Russell 2000; Tu et al. 2005). Competent cells were suspended in ice-cold fresh 100 mM CaCl2 solution.
Transformation and gene expression
For transformation, nanoconjugates were mixed with 200 μL of freshly prepared competent cells and incubated on ice. After 30 min, heat shock was given for 90 s at 42 °C, and instantly transferred on ice for 2 min, followed by addition of 800 μL of Luria–Bertani (LB) broth. Finally, cells were incubated at 37 °C for 1 h, followed by spreading 100 μL aliquots on nutrient agar plates containing 10 μg/mL ampicillin. Colonies grown after overnight incubation were counted. 200 μL of competent cells and 800 μL of Luria–Bertani broth were used as negative control, while an equal amount of plasmid DNA under similar conditions was used as a positive control.
Physicochemical characterization
The Fourier transform infrared spectroscopy (FTIR) spectra were recorded in diffused reflectance (DRS) mode using Perkin-Elmer D100 spectrophotometer with a resolution of 4 cm−1. The X-ray photoelectron spectroscopy analyses were performed on THERMO K-Alpha XPS machine, at a pressure better than 1 × 10−9 Torr. The core-level spectra for all the samples were recorded with un-monochromatized Mg Kα radiation (photon energy of 1253.6 eV) at pass energy of 50 eV, electron takeoff angle of 90° and overall resolution at 0.1 eV. All the X-ray photoelectron spectroscopic spectra were background-corrected using Shirley algorithm, and their core-level binding energies were aligned with respect to the adventitious C1s binding energy (BE) of 285 eV (Shirley 1972). Zeta (ζ) potential measurements were taken using Malvern 2000 Zetasizer.
Results and discussion
PEO20–PPO69–PEO20 has linear A–B–A architecture, wherein end of one segment is covalently joined to the head of the other segment. In PEO20–PPO69–PEO20, the PPO chain (hydrophobic) has potential to invade lipid bilayers, whereas PEO chain (hydrophilic) weakly adsorbs at the membrane surface. Thus, depending upon the length of PPO/PEO chains, the PEO–PPO–PEO copolymer can act either as a membrane sealant or as a permeabilizer. Furthermore, PEO can offer solubility and cover DNA–copolymer complex from immune recognition, which can protect DNA from degradation (Batrakova and Kabanov 2008; Gebhart and Kabanov 2003; Wang et al. 2012). However, the physicochemical aspects that govern the association of negatively charged plasmid DNA with PEO20–PPO69–PEO20 are not well understood; therefore, we have employed a number of sensitive techniques to understand these interactions.
The DNA compaction ability of PEO20–PPO69–PEO20 was evaluated by gel retardation assay using agarose gel electrophoresis, wherein an equal amount of DNA was loaded in each well (Fig. 1A). The increasing amounts of PEO20–PPO69–PEO20 with respect to plasmid DNA (lanes 2–8) led to increasing retardation of plasmid DNA mobility, since an increasing amount of plasmid DNA retained in the wells. In order to further understand this mechanism, samples were subjected to zeta potential measurements, which revealed a continuous reduction in negative zeta value of plasmid DNA from − 30.6 to − 10.8 mV with an increase in DNA-to-polymer ratio of 1:100 (Fig. 1B). Since PEO20–PPO69–PEO20 is a non-ionic polymer, the shift in the zeta potential value from highly negative toward neutral is indicative of increasing DNA sequestration, potentially through H-bonding interactions between the DNA and the polymer. The zeta potential measurements support the observations from gel migration assay, which suggested a reduction in DNA mobility, potentially due to a reduction in overall negative charge as a function of the increase in polymer concentration. Figure 1C shows a change in hydrodynamic diameter of plasmid DNA/PEO20–PPO69–PEO20 nanoconjugates. Interestingly, an increase in polymer concentration with respect to plasmid DNA led to dramatic reduction in diameter. This suggests that as the concentration of polymer is increased, either the number of micelles is increased at the cost of particle size, or due to the presence of larger amount of polymer, the micelles become more compact.
As demonstrated in Fig. 2 (Panel-1) pure plasmid DNA displays characteristic vibrational frequencies of DNA in the 1700–1500 cm−1 region and phosphate (PO43−) stretching frequencies in 1250–1050 cm−1 (curve 2). Two strong absorption bands at 1230 and 1088 cm−1 (marked*) are assigned to asymmetric and symmetric stretching vibrations of PO43− group, respectively. Vibrational frequencies at 1701, 1661, 1610, 1582 (shoulder), 1534 and 1482 cm−1 are assigned to guanine (C7=N stretching), thymine (C2=O stretching), adenine (C7=N stretching), purine stretching (N7), in-plane vibration of cytosine and guanine and in-plane vibration of cytosine, respectively. Additionally, C–C deoxyribose stretching was observed at 969 cm−1, while deoxyribose B-marker frequency was observed at 875 cm−1. The detected plasmid DNA frequencies are in agreement with the published literature (Choosakoonkriang et al. 2003; Taillandier and Liquier 1992). Comparative analysis of spectra provided evidence for interaction between the PO43− groups of plasmid DNA and PEO20–PPO69–PEO20. In pristine PEO20–PPO69–PEO20 (curve 1), there were no vibrational frequencies between 1700 and 1500 cm−1 region, but the signatures appeared in the nanoconjugates without any significant changes (curves 3 and 4). As displayed in curves 3 and 4 (plasmid DNA/polymer, 1:1 and 1:10, respectively), after interaction, shifts in vibrational frequencies were observed in both the asymmetric and symmetric stretching of PO43− from 1230 to 1237 (1:1) and 1242 (1:10) cm−1, and from 1088 to 1118 cm−1, respectively. However, there were no considerable changes in other functional groups; thus, Fourier transform infrared spectroscopy provided strong confirmation that PO43− of plasmid DNA interacts with PEO20–PPO69–PEO20 during nanoconjugates formation.
As illustrated in Fig. 2 (Panel-2A), core-level X-ray photoelectron spectroscopy spectra of C1s recorded for plasmid DNA can be deconvoluted into three energy levels. Binding energies (BE) maxima at 285.0, 286.4 and 287.9 eV can be assigned to the aromatic carbon chain, carbon attached to O and N species, and C=O present in nitrogenous bases, correspondingly. The core-level X-ray photoelectron spectroscopy spectrum of P2p (Fig. 2, Panel-2B) arising from plasmid DNA shows binding energy maximum at 133.4 eV, and it can be specified to PO43− groups of plasmid DNA. Figure 2, Panel-2C, shows X-ray photoelectron spectroscopy spectrum of N1s core level in plasmid DNA, which can be deconvoluted into two energy levels, in which the lower binding energy component at 399.2 eV can be assigned to the –C=N group, whereas the higher binding energy feature at 400.4 eV can be devoted to –NH2. Similarly, the O1s recorded for plasmid DNA can be disintegrated into two energy levels at 531.3 and 532.75 eV maxima, which can be allotted to phosphate-oxygen and deoxyribose sugar-oxygen, respectively. Further demonstrated in Fig. 2 (Panel-2E–F) are X-ray photoelectron spectroscopy spectra of C1s and O1s present in PEO20–PPO69–PEO20. The core-level C1s spectrum can be deconvoluted into two major energy levels. The lower binding energy at 285.0 eV is assigned to alkyl chain of PEO20–PPO69–PEO20, and the higher binding energy at 286.5 eV can be assigned to carbon attached to oxygen. Binding energy at 532.7 eV was observed for O1s core-level spectrum originating from PEO20–PPO69–PEO20. Since pristine PEO20–PPO69–PEO20 does not have nitrogen, N signatures were not detected. Nevertheless, N signatures appeared in plasmid DNA and polymer nanoconjugates, further confirming their association. Representative core-level X-ray photoelectron spectroscopy spectra of N1s (in plasmid DNA:PEO-PPO-PEO at 1:1 ratio) are illustrated in Fig. 2 (Panel-2G), which shows a single binding energy component at 399.6 eV. Furthermore, core-level X-ray photoelectron spectroscopy spectra of C1s (at 1:10 ratio) are presented in Fig. 2 (Panel-2H). It was interesting to observe that there were no significant changes in the binding energies of C1s, even after conjugation at different weight ratios. The C1s X-ray photoelectron spectroscopy spectrum recorded for plasmid DNA:PEO–PPO–PEO (1:10 ratio) can be deconvoluted into two energy levels. The lower binding energy is observed at 285.0 eV, and the higher binding energy is recorded at 286.5 eV, which are comparable to PEO20–PPO69–PEO20.
Coexisting N and P are indicators of DNA since their presence is typically unaffected by surface contaminations. Still, in DNA nucleotides, N atoms are present in higher amount than P, and N has a higher X-ray photoelectron spectroscopy cross section. Therefore, between P and N, N can provide a more reliable reference for composition measurements (Petrovykh et al. 2003). Based on X-ray photoelectron spectroscopy analysis as represented in Table 1, it is noteworthy that in nanoconjugates, with the increasing concentration of polymer (with respect to plasmid DNA), C/N ratio increased significantly. The constant increase in C/N ratio indicates that the plasmid DNA is efficiently encapsulated in nanoconjugates.
Once the chemical interactions were confirmed, the nanoconjugates were tested for their proof-of-concept applicability in bacterial gene transformation. Ampicillin was used for the selection of transformation-positive bacteria during transformation because Escherichia coli DH5α originally did not has an ampicillin-resistant gene; therefore, only those cells that had taken up the foreign plasmid DNA (containing an ampicillin-resistant gene) could survive on the antibiotic containing plates. It is notable that only a few competent bacterial cells were able to uptake freely available plasmid DNA (without PEO20–PPO69–PEO20) as some bacterial colonies were grown on antibiotic containing plates (positive control at 1:0 ratio (Fig. 3A). Conversely, competent bacterial cells themselves could not survive on ampicillin plates due to the lack of the resistant gene and no colonies were developed in the negative control.
Transformation with nanoconjugates revealed that with the increasing ratio of PEO20–PPO69–PEO20 (with respect to plasmid DNA), the number of transformed colonies exhibited steep rise up to 1:10 weight ratios and reached the maximum. Further increment in the polymer concentration showed a significant decline till the evaluated ratios. Therefore, at 1:10 ratio transformation was maximum, and in comparison with pure plasmid DNA, it showed over sixfold higher transformation. Besides, gene expression is also a vital phenomenon by which information from a gene can be utilized to synthesize a functional product and it is essential to protect DNA from degradation during delivery. Therefore, the capability of transformed plasmid DNA to retain its functional integrity, i.e., it can synthesize gene product, is also demonstrated in cellular environment. After transformation, if plasmid DNA is delivered into cells and stays functional, green fluorescent protein (GFP) will be synthesized in the cells through the expression of gene. Figure 3B–E exhibits fluorescence originating from transformed bacterial colonies through the expression of green fluorescent protein gene, thus confirming the integrity and functionality of plasmid DNA in the cellular environment.
Conclusion
The present work provides insights into the physicochemical aspects that direct a PEO20–PPO69–PEO20 block copolymer to formulate nanoconjugates with plasmid DNA. Detailed analysis of these nanoconjugates revealed that the PO43− group of plasmid DNA is primarily involved in the interaction with the block copolymer. The size of nanoconjugates depends on the concentration of PEO20–PPO69–PEO20 with respect to plasmid DNA. Further, it is established that in the presence of PEO20–PPO69–PEO20, the plasmid DNA condenses and it can be efficiently transported inside the cells. Moreover, it was confirmed by the expression of genes within the cellular environment that the integrity and functionality of plasmid DNA was preserved in nanoconjugates. The vast molecular diversity of block copolymers offers new opportunities in fine-tuning their physicochemical properties to obtain nanoconjugates for a variety of applications with reduced toxicity.
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Acknowledgements
HKD gratefully acknowledges Government of India for National Overseas Scholarship and Japan Science and Technology (JST) Agency, Japan, for Sakura Science Asia Youth Exchange Fellowship. SS gratefully acknowledges Australian Government for an Endeavour Postdoctoral Research Award. VB acknowledges the Australian Research Council for a Future Fellowship (FT140101285) and Ian Potter Foundation for establishing Sir Ian Potter NanoBioSensing Facility at RMIT University. Authors acknowledge the support of RMIT node of AMMRF for providing technical assistance and access to characterization facilities.
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Daima, H.K., Shankar, S., Anderson, A. et al. Complexation of plasmid DNA and poly(ethylene oxide)/poly(propylene oxide) polymers for safe gene delivery. Environ Chem Lett 16, 1457–1462 (2018). https://doi.org/10.1007/s10311-018-0756-1
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DOI: https://doi.org/10.1007/s10311-018-0756-1