Article pubs.acs.org/JACS
Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells Shuai Liu,†,# Dezhong Zhou,†,# Jixiang Yang,† Hao Zhou,‡ Jiatong Chen,‡ and Tianying Guo*,† †
Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry, College of Chemistry, Nankai University, Tianjin 300071, China ‡ Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China S Supporting Information *
ABSTRACT: To transform common low-molecular-weight (LMW) cationic polymers, such as polyethylenimine (PEI), to highly efficient gene vectors would be of great significance but remains challenging. Because LMW cationic polymers perform far less efficiently than their high-molecular-weight counterparts, mainly due to weaker nucleic acid encapsulation, herein we report the design and synthesis of a dipicolylamine-based disulfide-containing zinc(II) coordinative module (ZnDDAC), which is used to functionalize LMW PEI (Mw ≈ 1800 Da) to give a non-viral vector (Zn-PD) with high efficiency and safety in primary and stem cells. Given its high phosphate binding affinity, Zn-DDAC can significantly promote the DNA packaging functionality of PEI1.8k and improve the cellular uptake of formulated polyplexes, which is particularly critical for hard-to-transfect cell types. Furthermore, Zn-PD polymer can be cleaved by glutathione in cytoplasm to facilitate DNA release post internalization and diminish the cytotoxicity. Consequently, the optimal Zn-PD mediates 1−2 orders of magnitude higher gluciferase activity than commercial transfection reagents, Xfect and PEI25k, across diverse cell types, including primary and stem cells. Our findings provide a valuable insight into the exploitation of LMW cationic polymers for gene delivery and demonstrate great promise for the development of next-generation non-viral vectors for clinically viable gene therapy.
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INTRODUCTION Genetic engineering has fueled tremendous interest in numerous biological processes for delivering nucleic acids to modulate specific protein expression.1−3 Current gene and cell therapies generally depend on direct in vivo transfection of pathogenic tissues or ex vivo manipulation of cells prior to transplantation.4,5 Primary cells are freshly isolated from organisms, and delivering specific gene codings to these nonproliferating cell types (e.g., hepatocytes and neurons) can facilitate treatment of numerous related diseases, such as Parkinson’s disease.6,7 Alternatively, ex vivo manipulation of stem cells via secretion of a broad spectrum of neurotrophic and angiogenic growth factors can induce tissue regeneration and benefit biological processes.8,9 However, to date, these cells have been notoriously difficult to transfect,10−12 and therefore, far more efficient gene vehicles are in imperative demand, focusing on these non-proliferating cell types of interest rather than those dividing cell lines. Non-viral vectors, due to their scalable production, nonpathogenicity, and structural flexibility,13−16 have presented promising alternatives to viral vehicles that are associated with safety concerns.17−20 Thereunto, the cationic polymer family © XXXX American Chemical Society
has emerged as the major class of non-viral carriers; however, their translation to the clinic has been severely hampered because of their high cytotoxicity and polyplex instability in serum.15,21,22 Previously, Zhou et al.15 circumvented these obstacles by designing poly(amine-co-ester) terpolymers with low positive charge density; moreover, peptides with negative charge were coated on the formulated polyplexes. Indeed, this is an innovative attempt toward tuning the balance of gene delivery efficiency and cytotoxicity. Nonetheless, besides a new elaborate design and construction of gene delivery systems, to face and break through the intrinsic drawbacks of traditional cationic polymers would be of great significance and would further open up opportunities for polycation vectors, especially given their advantages of scalability and ease of modification. To this end, low-molecular-weight (LMW) cationic polymers stand out because they are relatively well tolerated, whereas they largely lag behind in efficacy compared to their highmolecular-weight (HMW) counterparts.23,24 To navigate this obstacle, Dahlman et al.23 developed LMW polyethylenimine Received: December 28, 2016 Published: March 21, 2017 A
DOI: 10.1021/jacs.6b13337 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
Figure 1. Design of Zn-PD nonviral vector for transfection of primary and stem cells. (a) Chemical structure and synthesis of DDAC ligand and ZnPD polymer. (b) Zn-PD condenses DNA tightly, and the formulated polyplex shows robust cellular uptake, both attributed to high affinity between Zn coordinative ligand and phosphorylated components. Once into the cytoplasm, DNA is released via GSH-triggered polyplex disassociation, facilitating high transfection efficiency of primary and stem cells.
purpose, metal coordination, particularly that of zinc dipicolylamine (Zn-DPA) analogues, may provide an option for DNA binding because of its high affinity to phosphodiester moieties.28,29 Moreover, to the best of our knowledge, no Zn-DPA analogue has been used for plasmid DNA delivery thus far. Additionally, as phosphate-containing components largely exist on cell membranes, we envisage that introducing the function of the Zn-coordinative ligand to LMW PEI would not only tremendously strengthen its DNA binding affinity but also drastically enhance the cellular uptake of the LMW PEIbased polyplexes, which would be particularly beneficial for transfecting primary and stem cells. Tight DNA binding also signifies difficult DNA unpacking post cell entry. Nevertheless, spatiotemporally controllable DNA release, exactly and at the right time, into cytoplasm is one of the prerequisites for efficient gene transfection.25,30 To this end, stimuli-responsive components like disulfide bonds provide a convenient pathway.31,32 Through disulfide linkage
(PEI) modified with alkyl chains to deliver siRNA with high efficacy. However, such small molecules with delicate structure, utilized to deliver siRNA, are not capable of condensing large plasmid DNA tightly and inducing highly efficient DNA expression. It is known that delivering specific DNA to cells and tissues plays a critical role in treating various inherited or acquired diseases and defective tissue regeneration, contributing to its broad factor secretion, long-term gene expression, and the source therapy of diseases.25,26 Nonetheless, to date, plasmid DNA delivery for stable protein expression utilizing functionalized LMW PEI has been far from satisfactory. LMW cationic polymers, as gene carriers, show performance inferior to that of their HMW counterparts, and this hurdle is attributed to the lack of multivalency for tight DNA binding.27 Therefore, it is conceivable that improving the DNA packaging functionality of LMW PEI would significantly enhance the transfection efficiency, making it even higher than that of the commercial reagent PEI25k, without extra cytotoxicity. For this B
DOI: 10.1021/jacs.6b13337 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Article
Journal of the American Chemical Society
Figure 2. Zn-DDAC functionalization matters for superior biophysical performance. (a) Electrophoretic retardation analysis of polymer/DNA polyplexes at different weight ratios (0.01, 0.05, 0.1, 0.2, and 0.3). (b) DNA release assays in the absence (0 mM) or presence (10 mM) of GSH. The weight ratio of polymer/DNA is fixed at 10:1. (c) Zn-PD2 and Zn-PD4 binding with DNA show sizes 150−200 nm, much smaller than the PEI1.8k and PD2 counterparts. *P < 0.05 indicates decreased size compared to PEI1.8k group. (d) Zn-PD2/DNA and Zn-PD4/DNA polyplexes exhibit low zeta potentials in comparison with the PEI1.8k counterpart. *P < 0.05 indicates decreased zeta potential compared to PEI1.8k group. (e) Adsorption behavior of 293T cells to naked gold electrodes and Zn-DDAC- and DDAC-modified electrodes. (f) Zn-DDAC shows substantially higher viability than DDAC. *P < 0.05 indicates superior cell viability compared to DDAC.
between Zn-coordinative functional ligands and LMW PEI, first the resultant polymer can be cleaved by glutathione (GSH) existing in the cytosol to generate LMW PEI and Zncoordinative monomer inside cells, where neither could condense DNA tightly enough, leading to fast and accurate release of DNA into the cytosol; second, GSH-triggered degradation preserves the inherent low cytotoxicity of LMW PEI. Herein, we report the design and synthesis of a disulfidecontaining Zn(II)-coordinative DPA analogue module (ZnDDAC), followed by functionalizing PEI1.8k to give a highly effective and safe non-viral vector (Zn-PD). This strategy could achieve the transformation of common and easily obtained LMW PEI to a highly efficient and safe gene vehicle. The GSHtriggered reduction was designed to facilitate DNA release and reduce cytotoxicity. The internalization mechanism of this newfashioned gene delivery system is discussed in detail. Transfection efficiency is evaluated across conventional cell lines (293T, HCT116, and Hela) and special cell types, including myeloma cells (SP2/0), primary cells (SC), and stem cells (rBMSC, hBMSC, and rADSC). The unique binding affinity with phospholipid cell membrane makes Zn-PD highly appealing to transduce diverse cells, especially those specific but hard-to-transfect cell types. Our strategy offers a practical and versatile platform to functionalize LMW cationic polymers for high-performance primary and stem cell gene transfection. More importantly, the components in Zn-PD polymers are not immutable, and taking into account the numerous metal coordination architectures, massive cationic polymers, and diverse stimuli-response linkages, the system proposed here holds huge promise and can further inspire researchers in the field of non-viral gene delivery.
polymers. To this end, GSH-cleavable disulfide bonds were incorporated between LMW PEI and the Zn-coordinative group to facilitate DNA unpacking. As outlined in Figure 1, a disulfide-containing coordinative ligand, DDAC, was designed, and the peaks at 5.6 and 6.2 ppm, assigned to vinyl groups, indicated its successful synthesis (Figures S1−S3). Afterward, DDAC was Zn-coordinated and functionalized on branched PEI1.8k through Michael addition reaction, giving Zn-PDm polymers, where “m” represents the number of Zn-DDACs on each PEI1.8k molecule. As shown in Figure S4, when m > 4, excess unreacted Zn-DDAC remained because of the sterichindrance effect. Unreducible Zn-PH2 polymer without disulfide bonds was prepared as control (Figure S5). Consequently, Zn-PD0.5, Zn-PD1, Zn-PD2, Zn-PH2, and Zn-PD4 were further investigated in gene transfection experiments. Zn-DDAC Functionalization Improves Transfection Efficiency of LMW PEI. Zn-DDAC ligand functionalization is expected to improve the DNA packaging functionality of LMW PEI and enhance the interaction between polymers and cell membranes. As shown in Figure 2a, Zn-PD2 condensed DNA completely at an extremely low weight ratio of 0.2, while for PEI1.8k, the weight ratio increased to 1 (Figure S6). As expected, PD2 without Zn coordination exhibited weak DNA packaging ability comparable to PEI1.8k, further demonstrating the critical role Zn coordination played in promoting the DNA binding affinity. In addition, disulfide linkages can be cleaved in cytosol by GSH, which exists in intracellular compartments at high concentration of 1−10 mM, while the polyplexes maintains stable due to low concentration of GSH (1−10 μM) in extracellular environment.36,37 Thus, Zn-PD2/DNA polyplex could be efficiently disassociated to release DNA by GSH cleavage; in contrast, Zn-PH2 without disulfide bonds still condensed DNA tightly in the presence of GSH (Figure 2b). Zn-DDAC modification decreased the cationic effective mass of PEI1.8k moiety at equivalent w/w ratios, but even at equivalent N/P ratio, it was Zn-PD2 rather than PEI1.8k that could release DNA from their corresponding polyplexes in the presence of
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RESULTS AND DISCUSSION Design and Synthesis of Zn-PD Polymers. Although DPA-based Zn-coordinative ligands showed high phosphodiester moiety binding,33−35 the structure should still be well tailored and optimized to promote the functionality of the C
DOI: 10.1021/jacs.6b13337 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
Figure 3. Zn-PD polymers show high transfection efficiency and low cytotoxicity in 293T cells. (a) Gluciferase activity and (b) cytotoxicity of 293T cells post transfection. Zn-PD2 and Zn-PD4 exhibit significantly higher transfection efficiency than control PEI1.8k, Zn-PH2, and commercial transfection reagents Xfect and PEI25k. *P < 0.05 indicates superior gluciferase activity compared to PEI1.8k (n = 4). (c) EGFP transfection efficiency in 293T cells with the weight ratio of polymer/DNA fixed at 10:1. Commercial Xfect and PEI25k are used as per protocols.
GSH (Figure S7). These results indicate that Zn-DDAC module functionalization can significantly improve the DNA packaging functionality of LMW PEI, which is also capable of leading to DNA release by GSH-triggered biodegradability. Although fully packaged, Zn-PD2/DNA and Zn-PD4/DNA polyplexes both exhibited particle sizes below 200 nm, just suitable for cellular uptake (Figure 2c). In contrast, for the PEI1.8k counterpart, sizes increased to 270−480 nm, and this might be due to relatively looser DNA packaging functionality, which was consistent with above gel electrophoresis assays (Figure 2a). Moreover, unlike PEI1.8k/DNA polyplexes, ZnPD2 and Zn-PD4 counterparts showed great stability after 2 h incubation (Figure S8). Besides this advantage, Zn-DDAC functionalization also reduced the cationic effective mass of PEI1.8k moiety at equivalent polymer/DNA weight ratios, resulting in low zeta potentials of the Zn-PD-based polyplexes (Figure 2d). At w/w ratios of 5:1 to 20:1, Zn-PD4/DNA polyplexes exhibited zeta potentials only 0−5 mV, much lower than the PEI1.8k counterpart of 8−17 mV. The low zeta potentials can greatly benefit the serum stability of polyplexes and decrease the cytotoxicity, and this is particularly significant for those fragile primary and stem cells. Since that Zn-DDAC ligand shows high binding affinity to phosphate components, strong interactions between phospholipid cell membranes and Zn-coordinated ligands are expected. DDAC and Zn-DDAC were immobilized on the quartz crystal microbalance (QCM) gold electrode via Au−S bond formation. As shown in Figure 2e, adsorption behavior of 293T cells to Zn-DDAC was studied using QCM. As the mass change on the electrode surface is proportional to the output oscillation frequency shift, the frequency shift, attributed to interactions between gold electrode and cells, reached 29 Hz after injection of 293T cells. However, the frequency shift of DDAC-modified electrode decreased to 23 Hz because of the spatial repulsive effect post cell adsorption. In contrast, after Zn coordination,
the frequency shift increased largely to 32 Hz, indicating significant cell affinity enhancement of DDAC by Zn coordination. This is the first direct quantification using QCM to confirm that Zn coordination can improve the interaction between ligands and cells, instead of previous indirect experimental evidence, such as enhanced cellular uptake efficiency.38 Additionally, Zn coordination could greatly diminish the cytotoxicity of pyridine-containing ligands (Figure 2f), attributed to avoidance of direct cell touching by pyridyl groups. Gene transfection was first evaluated on 293T cells, and commercial transfection reagents Xfect and PEI25k were used as positive controls. As shown in Figure 3a, Zn-DDAC functionalization demonstrated its superiority in benefiting the gene delivery. At w/w = 10, up to 16-, 237-, and 289-fold higher gluciferase activity was observed with Zn-PD1, Zn-PD2, and Zn-PD4, respectively, compared to PEI1.8k. Furthermore, the optimal Zn-PD4 still mediated 3.5- and 6.0-fold enhancements of gluciferase activity compared to the commercial Xfect and PEI25k, respectively. GFP images post transfection further validated that Zn-PD2 and Zn-PD4 outperformed the commercial reagents (Figure 3c). Meanwhile, Zn-PD/DNA polyplexes preserved high cell viability, while unreducible ZnPH2/DNA showed high cytotoxicity and low transfection efficiency (Figure 3a,b), which might be attributed to difficult DNA release from the polyplexes. In addition, PD2 and ZnPD2 were evaluated on gene transfection, and Figure S9 showed that Zn-PD2 mediated 3 orders of magnitude higher gluciferase activity than PD2 with much lower cytotoxicity, further verifying the significance of Zn coordination on gene transfection improvement. These results demonstrate that ZnDDAC ligand modifying on LMW PEI dramatically promotes the DNA binding affinity, and once into intracellular environment, GSH-triggered reduction contributes to the DNA release and low cytotoxicity. These features together impart Zn-PD D
DOI: 10.1021/jacs.6b13337 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
Figure 4. Zn-PD4 outperforms commercial Xfect and PEI25k at low DNA doses and high serum concentrations. (a,b) Transfection efficiency of ZnPD4, Xfect, and PEI25k at different DNA doses in 293T cells. Serum concentration is fixed at 10%. (c,d) Transfection efficiency at different serum concentrations in 293T cells. DNA dose is fixed at 1 μg/well. The weight ratio of Zn-PD4/DNA polyplex is fixed at 10:1. Commercial Xfect and PEI25k are used as per protocols. Error bars represent the standard deviation (n = 4). *P < 0.05 indicates superior gluciferase activity compared to Xfect group.
Mechanistic Studies. The vector plays a pivotal role in gene medicine; however, little is known about the process and mechanism of gene delivery.18 The detailed polyplex cellular uptake, DNA release, and polyplex internalization pathway need further definition. To look into the reason behind robust transfection efficiency of Zn-PD polymers, the cellular uptake and DNA release behavior of PEI1.8k/DNA, Zn-PH4/DNA, and Zn-PD4/DNA polyplexes were compared. As shown in Figure 5a, numerous Zn-PH4/DNA and Zn-PD4/DNA polyplexes were accumulating inside the cells, confirming that Zn coordinative functionalization significantly improved the cellular uptake efficiency of polyplexes. Furthermore, FITClabeled Zn-PH4 and Cy3-labeled DNA exhibited co-localization because of difficult DNA release from polyplexes, in accordance with our previous prediction. In contrast, the majority of DNA was efficiently released from the Zn-PD4/DNA polyplex and accumulated around the nucleus, while the small PEI residue might have been rapidly cleared to the extracellular environment. These results were supported by the reduced green fluorescence and enhanced red fluorescence with the increase of incubation time (Figure S10). In addition, cellular uptake of Zn-PD4/DNA and PEI25k/DNA polyplexes for 1 h treatment was compared. Each cell seemed to have polyplexes inside; however, much more Cy3-DNA and dramatically stronger red fluorescence were observed in Zn-PD4/DNA-treated cells compared to the PEI25k counterpart (Figure S11). This highlights the superiority of Zn-PD4 that has high affinity with cell membranes, facilitating cellular uptake and benefiting ultimately gene transfection. The internalization pathway is a basic parameter for a gene delivery system, and that of DPA-based zinc(II) coordination needs further confirmation. To verify whether endocytosis is
polymer as an excellent gene vector, hopefully achieving robust gene transfection in those significant but hard-to-transfect cell types, such as primary and stem cells. Superiority of Zn-PD4 at Low DNA Doses and Serum Resistance. For clinical applications, low DNA doses and serum resistance have been two significant parameters.3,39 Lower DNA dosage can reduce the cytotoxicity and minimize the side effects. Aside from this, serum stability also plays an important role in in vivo gene transfection. Transfection efficacy of Zn-PD4 as well as commercial Xfect and PEI25k at low DNA doses was first evaluated. The traditional transfection usually uses a DNA dose of 1−2 μg/well for 24-well plate. As shown in Figure 4a,b, Zn-PD4 exhibited a high efficiency at low DNA doses of 0.4 and 0.2 μg, greatly outperforming commercial Xfect and PEI25k, which showed sharply decreased efficiencies at decreasing DNA doses. The high efficacy of Zn-PD4 at low DNA doses is attributed to the high affinity between Zncoordinated polyplexes and phospholipid cell membranes. Afterward, the serum resistance was studied by transfection in media containing 10−50% serum. Zn-PD4 preserved massive transfection efficiency in the presence of 10−50% serum, conversely, much lower efficacy was observed for Xfect and PEI25k in high serum concentrations (Figure 4c,d). The excellent serum resistance functionality of Zn-PD4/DNA polyplexes is attributed to the several anti-protein hydroxyl groups in the side chain of Zn-PD4 and integral low polyplex zeta potential (Figure 2f), which can be explained on the basis that the DDAC module functionalization significantly decreased the cationic effective mass of PEI1.8k moiety at equivalent w/w ratios. These encouraging results reveal the great potential of Zn-PD4 for further in vivo applications. E
DOI: 10.1021/jacs.6b13337 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society
Figure 5. Mechanistic studies of internalization. (a) Cellular uptake images of HCT116 cells treated with FITC-PEI1.8k/Cy3-DNA, FITC-Zn-PH4/ Cy3-DNA, and FITC-Zn-PD4/Cy3-DNA polyplexes for 4 h. The cell nuclei are stained with DAPI. The arrows indicate the DNA release from polyplexes and accumulation around nuclei. (b) The internalization of Zn-PD4/DNA polyplexes incubated at 37 or 4 °C. *P < 0.05 indicates better inhibition compared to 37 °C. (c) Cellular uptake of Zn-PD4/DNA polyplexes in the presence of various inhibitors. *P < 0.05 indicates better inhibition compared to standard Zn-PD4/DNA polyplex. (d) Gluciferase activity of Zn-PD4/DNA polyplexes in the presence of various inhibitors. *P < 0.05 indicates better inhibition compared to standard Zn-PD4/DNA polyplex. The weight ratio of all polymer/DNA is fixed at 10:1.
respectively. Sharply differently, the efficacy diminished to 42% with MβCD preincubation. From these results, we can conclude that Zn-PD polymers formulated with DNA mediates ultrahigh cellular uptake efficacy, which is mainly facilitated by CIE pathway. Different from PEI1.8k/DNA polyplexes, Zn-PD4 counterparts exhibited suitable sizes for clathrin-mediated endocytosis (
