Bioreducible Gene Delivery Vector Capable of Self-Scavenging the

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Bioreducible Gene Delivery Vector Capable of Self-Scavenging the Intracellular Generated ROS Exhibiting High Gene Transfection Long-Hai Wang, Ting Wu, De-Cheng Wu, and Ye-Zi You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04327 • Publication Date (Web): 15 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016

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ACS Applied Materials & Interfaces

Bioreducible Gene Delivery Vector Capable of SelfScavenging the Intracellular Generated ROS Exhibiting High Gene Transfection Long-Hai Wang†, Ting Wu†, De-Cheng Wu‡*, Ye-Zi You†* † Key Laboratory of Soft Matter Chemistry, Chinese Academy of Science, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China. ‡ Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. KEYWORDS: gene delivery, self-assemble, bioreducible nanomicelles, reactive oxygen species, self-scavenge

ABSTRACT. Cationic polymer vectors have received increasing attention for gene delivery in biotechnology over the past two decades, but few polymer vectors were used in clinical applications due to their low gene transfection efficacy. One of the major reasons is that the conventional cationic polymers can induce the increasing of intracellular reactive oxygen species (ROS) concentration and oxidative stress, which reduces the gene transfection efficacy. Here, we create a novel class of thioether dendron−branched polymer conjugate and self-assemble this

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conjugate into bioreducible cationic nanomicelles with disulfide bond connecting the thioether core to the cationic shell. The obtained nanomicelles have a unique ROS self-scavenging ability, thereby dramatically improving gene transfection efficacy.

Introduction Gene therapy offers tremendous promise for treating various human diseases including genetic disorders,1 cancers,2 viral infections,3 and cardiovascular diseases.4 The viral vectors can achieve high transfection efficiency in gene delivery, but the fatal defect for virus is their safety issue and complicated packaging process.5-7 In contrast, cationic polymers, as one kind of non-viral vectors, are much safer than viral vectors, and they are easy for synthesis and molecular structural design.8-10 However, the gene transfection efficacy using cationic polymers is still much lower than that of viral vectors until now, limiting their uses in medical applications. Therefore, at present time, the researches attempt to improve the gene transfection efficacy by designing new molecule structures with multi-functionalities. However, many chemical molecules and nanomaterials not only can induce the generation of some new molecules but also deplete some existing molecules in the cell after they enter cells, thereby substantially changing the intracellular physiological environment, which in return may have unexpected effects on the activity of protein and gene.11-13 For example, high doses of vitamin C can highly increase the level of intracellular ROS concentration and the generated ROS exerts antitumorigenic activity.14 Many nanoparticles (i.e. silver nanoparticles, titanium nanoparticles, titanium dioxide, zinc oxide nanoparticles, etc) have also been reported to increase ROS generation, leading to oxidative stress in the cell.11-13, 15 Cationic polymers binding electrostatically to negatively charged DNA form polyplex nanoparticles, which can deliver gene into target cell. However, in the cell, the interaction of these polyplex nanoparticles (even cationic polymers) with mitochondria could


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cause the mitochondrial damages, which could stimulate the production of excess intracellular ROS.15-20 In normal physiological environment, the intracellular ROS keeps at a low level, and the generated ROS can be neutralized by natural antioxidant defenses. However, once producing excess ROS, the antioxidant defenses will be invalid, leading to oxidative stress.11 Oxidative stress not only has great effect on the secretion of chemokines and cytokines, but also can alter mRNA expression, which reduces the target gene expression even at a moderate (i.e. noncytotoxic) oxidative stress.20-23 Most important, the generated ROS could also damage DNA if it was not scavenged in due time.14 Up to now, there is no effective delivery system that can timely self-remove the polyplex-generated ROS though it is very important for enhancing gene transfection efficacy. Here, we create a new nanomicelle gene delivery vector from thioether dendron−branched polymer conjugate. We have observed that the thioether cores of nanomicelles have unique ROS self-scavenging ability, and the cationic shells showing very strong interactions between DNA and cationic polymer, can fully condense the plasmid DNA at low N/P ratios. Therefore, formed polyplexes would transfect in normal intracellular homeostasis and achieve excellent gene transfection efficacy.

Results and discussion


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Figure 1. The formation of ROS self-scavenging gene delivery vector. (a) The scheme outlining the synthesis of thioether dendron-branched PEI conjugate. (b) The formation of nanomicelles and nanomicelles/DNA polyplexes. Thioether is ready to react with ROS including hydrogen peroxide and superoxide, forming sulfoxide or sulphone, and therefore, it can scavenge ROS. In order to investigate the scavenging of intracellular ROS generated by gene delivery vectors, we prepared thioether dendron, and linked the thioether dendrons to branched PEI via disulfide bonds as shown in Figure 1a. PEI (Mw, 25000 kDa) was first treated with Traut’s reagent, in situ producing reactive thiol onto PEI, subsequently, thioether dendron with a disulphide pyridine unit at the focal point (see the


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detailed procedure for the preparation of thioether dendron in Supporting Information) was added, the thiol unit of PEI immediately reacted with disulphide pyridine unit of thioether dendron via thiol-exchange, forming a novel class of thioether dendron-branched PEI conjugate with a glutathione-responsive disulfide linker (Figure 1a, PEI–SS–5D, each PEI macromolecule have been linked with 5 Dendrons). PEI–SS–5D was dissolved in chloroform, subsequently, the chloroform was removed by vacuum rotary evaporation to form a dry film. Water was added, and the mixture was sonicated, forming bioreducible nanomicelles (20 nm in diameter) with disulfide bond connecting the cationic shell to the thioether core as shown in Figure 1b. The timely scavenging of the intracellular generated ROS is very important for increasing gene transfection efficacy and the clinic safety of gene therapy. In order to check whether the prepared cationic nanomicelles with thioether core can readily scavenge ROS, the nanomicelles were treated with H2O2. In the FI-IR and XPS spectra (Figure 3a and 3b), it is very obvious that signals for S=O appeared, indicating that the prepared cationic nanomicelles with thioether core can scavenge H2O2. Moreover, the intracellular ROS was measured using DCFH-DA ROS Assay Kit. The cells under oxidative stress will be shown as green fluorescence from the oxidized DCF. As shown in Figure 3c, after the incubation for 24 h, the ROS level in cells treated with PEI/DNA polyplexes was similar to that of cells treated with 100 µM H2O2. Adding H2O2 into the incubation system further increases intracellular oxidative stress level, which highly decreased polyplex transfection as shown in Figure 3d. Here, though the prepared cationic nanomicelles/DNA polyplexes have much higher surface charge (Figure 2d) than PEI/DNA polyplexes, it is noteworthy that no obvious oxidative stress was observed even after 24 h’s incubation as shown in Figure 3c. This is due to that thioether dendron core can effectively self-


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scavenge the generated ROS, which is consistent with observations that thioether polymer can react with ROS.24

Figure 2. TEM images and DLS analysis of the formed nanomicelles (a) and nanomicelle/DNA polyplexes (b). (c) Ethidium bromide displacement titrations for PEI, PEI-SS-5D non-micelle and PEI-SS-5D nanomicelles. (d) Zeta potential of PEI/DNA and PEI-SS-5D nanomicelles/DNA polyplexes. Agarose gel electrophoresis analysis of the PEI-SS-5D nanomicelles/DNA polyplexes (e) and the PEI/DNA polyplexes (f).


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Mitochondrial membrane potential (∆Ψm) plays a critical role in maintaining mitochondria function. In normal physiological environment, ∆Ψm keeps at high value, but a loss of ∆Ψm will affect mitochondria function and increase the ROS in cytoplasm, which will decrease the gene transfection efficiency. JC-1 is a common fluorescent probe to measure the ∆Ψm, for a mitochondria with a high ∆Ψm, the fluorescent probe forms aggregates and shows as orange-red fluorescent, however, JC-1 exists as a monomer and shows as green fluorescentat at depolarized membrane potentials. As shown in Figure 3e, most of JC-1 in HeLa cells incubated with buffer for 24 h showed intense orange-red fluorescence while JC-1 in HeLa cells incubated with H2O2 of 100 µM and PEI/DNA polyplexes for 24 h showed similar intense green fluorescence, which indicates that the cells incubated with PEI/DNA polyplexes have similar low ∆Ψm as the cells incubated with H2O2. It is noteworthy that JC-1 in the cells incubated with the prepared nanomicelles/DNA polyplexes have similar orange-red fluorescence with that incubated with buffer, which indicates that thioether dendron can act as an efficient ROS-scavenger that could reduce the ROS and protect the mitochondria from depolarizing.

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Figure 3. (a) FT-IR spectra for thioether dendron before and after being treated with H2O2. (b) XPS spectra (S2p region) for PEI–thioether dendron conjugate nanomicelles before and after


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being treated with H2O2 (1 mM). (c) Intracellular ROS assay results via fluorescence microscope (HeLa cells were treated with different formulations for 24 h). (d) Luciferase transfection efficiency of PEI in HeLa cells with H2O2 (0 and 100 µM). (e) Mitochondrial Membrane Potential analysis results via fluorescence microscope (HeLa cells were treated with different formulations for 24 h). Besides that the prepared nanomicelles are efficient ROS-scavenger, they are also excellent binder for DNA. Higher charge density would have higher binding affinity towards DNA.25,26 Firstly, the binding affinities of nanomicelles and DNA were tested by ethidium bromide (EtBr) displacement assay. As shown in figure 2c, the binding affinity can be presented as a charge excess (CE50) value, and the CE50 values were represented as N/P ratios of vector/DNA required to reduce the fluorescence intensity of DNA/EtBr complex to 50%. A lower CE50 value means higher binding affinity, and an excellent vector should give a CE50 value below 1.0.

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golden standard for cationic polymer vectors, the branched PEI25 k has high charge density, but it shows low binding affinity for DNA (CE50 = 1.58, Figure 2c). It is noteworthy that the 20 nm PEI-SS-5D micelles exhibited excellent DNA binding ability (CE50 = 0.60, Figure 2c), which resulted from the very high surface charge of nanomicelles (+63 mV, Figure S4). And the PEISS-10D nanomicelles also show stronger DNA binding ability (CE50 = 0.82, Figure S5) than PEI25 k. Generally, the branched PEI25 k can fully condense plasmid DNA at an N/P ratio around 5 (Figure 2f). However, the 20 nm PEI-SS-5D micelles can fully condense plasmid DNA at a low N/P ratio of 1 (Figure 2e). And PEI-SS-5D nanomicelles/DNA polyplexes showed high stability in NaCl solution (Figure S7). Generally, polymer gene vectors with strong DNA binding ability can condense DNA into tight polyplexes, which protects the DNA from degradation.29 On the other hand, the efficient intracellular DNA release is necessary for obtaining high gene transfection efficiency,29 however, the formed tight polyplexes maybe unfavorable for efficient DNA release. Therefore, an excellent gene vector must balance sufficient binding affinity to condense DNA with the


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ability to release the loaded DNA. Here, the constructed nanomicelles contain cationic shells and thioether cores with disulfide bond linkages which can be cleaved by high concentration of intracellular glutathione (GSH). Then the nanomicelles will break up and disassemble, and the loaded DNA will be effectively released. As shown in Figure S6, in vitro experiment results showed that the nanomicelles with disulfide linkages could release the entrapped DNA after treating with GSH while the nanomicelles without disulfide linkages cannot. So the PEI–5D nanomicelles without disulfide linkages (Figure S10) showed lower gene transfection efficacy than PEI–SS–5D nanomicelles. To evaluate the effect of increased oxidative stress on gene transfection, PEI/DNA polyplexes were transfected in 293T cells in mediums with H2O2. Transfection efficiency highly decreased as the H2O2 was added (as shown in Figure 3d, Figure 4a). For comparison, we also prepared similar bioreducible nanomicelle with a fluorinated core (without ROS self-scavenging ability). The thioether core-containing nanomicelle/DNA polyplexes and fluorinated core containing nanomicelles/DNA polyplexes were transfected in 293T cells in mediums with H2O2. Transfection efficiency of the thioether core-containing nanomicelles/DNA polyplexes remained almost unchanged as the H2O2 was added, while transfection efficiency of fluorinated core containing nanomicelles/DNA polyplexes highly decreased (as shown in Figure 4a), indicating that the elevated intracellular ROS concentration will reduce the gene transfection activity and that timely and effectively removal of generated excess ROS by thioether core is very important for increasing gene transfection efficacy. Because excess ROS can affect the secretion of chemokines, cytokines and alter mRNA expression, which reduces the gene expression even at subtoxic concentrations.21,22 Lipofectamine 2000 was used as another vector to verify the effect of ROS on gene transfection (Figure S11). Lipofectamine 2000/DNA complexes were


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transfected in 293T cells in mediums with ROS inhibitor (GSH-OEt) or ROS inducer (H2O2). The results showed that an elevated level of ROS (medium with H2O2) would reduce the transfection efficiency and a depressed level of ROS (medium with GSH-OEt) would improve the transfection efficiency. To evaluate the gene transfection efficacies of nanomicelles complexed with different plasmid DNA in different cell lines. Nanomicelles with different amount of thioether dendrons were prepared and subsequently complexed with DNA. As shown in Figure 4b, these nanomicelles exhibit much higher GFP gene transfection efficacies compared with PEI25 k at N/P ratios of 5 and 10 in 293T cells. Furthermore, the PEI-SS-5D nanomicelles/DNA polyplex at an N/P ratio of 3 exhibit remarkably higher GFP gene transfection efficacy compared with PEI/DNA polyplex at an N/P ratio of 10 in 293T cells. And the CLSM showed that the PEI–SS–5D nanomicelles/DNA polyplex at an N/P ratio of 3 and PEI/DNA polyplex at an N/P ratio of 10 had no significant difference for cell uptake efficiency (Figure S8). Further luciferase gene transfections were carried out in 293T cells (Figure 4c), B16F10 cells (Figure 4d) and Hela cells (Figure 4e), and all results showed that PEI-SS-5D nanomicelles/DNA polyplexes exhibited higher gene transfection efficacy than PEI/DNA polyplexes. And the GFP gene transfection test with a medium containing 10% FBS was shown in Figure S9, the transfection efficacy of PEI–SS–5D nanomicelles/DNA polyplex at an N/P ratio of 2 remained unchanged as FBS added, but the transfection efficacies of PEI–SS–5D nanomicelles/DNA polyplex at an N/P ratio of 3 and PEI/DNA polyplex at an N/P ratio of 10 decreased as FBS added. In general, the cationic polymer vectors have much lower gene transfection efficiencies (

Bioreducible Gene Delivery Vector Capable of Self-Scavenging the

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