Bioreducible Cross-Linked Nanoshell Enhances Gene Transfection

Article pubs.acs.org/Biomac

Bioreducible Cross-Linked Nanoshell Enhances Gene Transfection of Polycation/DNA Polyplex in Vivo Ji-Gang Piao,† Sheng-Gang Ding,‡ Lu Yang,† Chun-Yan Hong,† and Ye-Zi You*,† †

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Jinzhai Road 96, Hefei 230026, Anhui, China ‡ Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei 230022, Anhui, P. R. China S Supporting Information *

ABSTRACT: In this study, we have prepared a self-cross-linking PEG-based branched polymer, which easily forms a bioreducible nanoshell around polyplexes of cationic polymer and DNA, simply via heating the polyplex dispersions in the presence of this self-crosslinking branched polymer. This nanoshell can prevent the polyplex from dissociation and aggregation in physiological fluids without inhibiting the electrostatic interactions between the polymer and DNA. Furthermore, glutathione (GSH) can act as a stimulus to open the nanoshell after it has entered the cell. The polyplexes coated with the bioreducible nanoshell show an obvious enhancement in gene transfection in vivo compared with bare polyplexes.

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INTRODUCTION Gene therapy is a promising method to treat various human diseases including cardiovascular diseases, viral infections, cancers, and genetic disorders.1 Successful gene therapy relies on efficient gene vectors. 2−4 Early clinical trials using recombinant viral vectors have reported significant problems, such as short-term transgene expression, an inability to persist in host cells, toxicity, and induced cancer.5,6 In response to these observed problems, a broad range of nonviral systems for gene delivery has been developed. The major categories of nonviral vectors include cationic lipids and cationic polymers.7,8 Cationic polymer vectors have gained increasing attention because of the ease with which they can be synthesized and structurally modified for specific biomedical applications.9 Cationic polymer systems deliver genes by forming condensed complexes with negatively charged deoxyribonucleic acid (DNA) through electrostatic interactions. Complexation with a cationic polymer protects the DNA from degradation and facilitates its cellular uptake and intracellular trafficking into the nucleus.10,11 However, the as-formed polyplexes tend to break apart or aggregate in physiological fluids, which contain serum components and salts.8,9 Copolymers of poly(ethylene glycol) (PEG) and the polycation, forming a PEGylated polyplex, have a longer circulation time because of their decreased tendency toward dissociation, aggregation, or nonspecific interactions with various biological macromolecules such as proteins.12−16 Although PEGylation appears to enhance the transfection efficiency in vitro,13 recent research shows that PEG interferes with polyplex formation; the electrostatic interactions between polymer and DNA are generally inhibited by the PEG moiety,17 leading to weaker binding. The weaker binding between the polyplex components may yield less compact structures and a less efficient protection against nucleases in vivo. Furthermore, © 2014 American Chemical Society

Müllen et al. have found that substitution of poly-L-lysine-amino sites with PEG caused the polyplexes to degrade more quickly, even at higher N/P ratios compared with nonmodified polymers.18 Bickel et al. have found that although PEGylated poly(ethylene imine) (PEI) demonstrates a slower uptake into the organs compared with 25 kDa PEI due to the shielding effect of PEG, the PEGylated polymer did not exhibit better stability in circulation and did not protect DNA from degradation.17 An optimal method to enhance gene transfection would therefore be a bioreducible PEG-based nanoshell that can isolate the polyplex from components in physiological fluids outside the cell while responding to a stimulus inside the cell to open the nanoshell and liberate the polyplex. Presently, to coat a PEGbased bioreducible nanoshell onto polyplexes is difficult, although coating solid nanoparticles with a PEG-based nanoshell is relatively easy.11,19 In this study, we have prepared a self-crosslinking PEG-based hyperbranched polymer, which easily forms a bioreducible nanoshell onto polyplexes simply via heating the polymer in the presence of the polyplex dispersions. This bioreducible nanoshell isolates the polyplex from serum components, salts, and other physiological fluid components, preventing the polyplex from dissociation and aggregation in blood fluid. The PEG moieties do not inhibit the electrostatic interactions between polymer and DNA or interfere with the formation of the polyplex; moreover, this nanoshell opens when inside the cell in response to GSH acting as a stimulus. The nanoshell-protected polyplexes exhibit high gene transfection, low hemolysis, low protein absorption, and low cytotoxicity. Received: April 8, 2014 Revised: June 10, 2014 Published: June 25, 2014 2907

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PEG(10), and PEI/DNA@PEG(20)). After being vortexed for 5 s, the temperature of the system was increased to 50 °C, and the system was incubated at this temperature for 15 min. In Vitro Transfection. Transfection efficiency with gWiz-Luc plasmid DNA tested in HEK-293T cell lines was measured as follows: cells were seeded at a density of 30 000 cells per well on 48-well cell culture plates, 24 h prior to the transfection experiment. The polyplexes were prepared as previously described, and 25 μL solution was added to each well containing 150 μL of DMEM with or without serum. After 4 h of incubation, the transfection mixture was removed and the cells were cultured for an additional 24 h in fresh DMEM. To determine the level of luciferase expression, the cells were incubated for 30 min at room temperature in 100 μL of cell lysis reagent buffer (Promega), following which the culture medium was discarded and cell lysate was harvested. The luciferase content of the cell lysate was then measured by injecting 100 μL of luciferase assay buffer (20 mM glycylglycine (pH 8), 1 mM MgCl2, 0.1 mM EDTA, 3.5 mM DTT, 0.5 mM ATP, 0.27 mM coenzyme) into 20 μL of cell lysate and integrating the luminescence over 10 s using single-tube Sirius luminometer. The total cellular protein in the cell lysate was determined by the bicinchoninic acid (BCA) protein assay using a calibration curve constructed with standard bovine serum albumin solutions (Pierce). The luciferase transfection results are expressed as relative light units (RLU) per milligram of cellular protein. Unless otherwise stated, the results are expressed as mean RLU/mg of protein ± SD of triplicate experiments. In Vivo Transfection. Animals were injected with complexes (20 μg DNA, 200 μL/mouse) through the tail vein. Organs including lung, liver, spleen, heart, and kidneys were dissected 48 h after the injection. Tissue homogenate was prepared by adding lysis buffer to the collected organs and homogenizing each organ for 15−20 s at 20 000 rpm for 10 min at 12 000 rpm. The tissue homogenate was then centrifuged in a microcentrifuge, and an aliquot of the supernatant (50 μL) was used for luciferase activity assay. The protein concentration of each tissue extract was determined by a standard protein assay. Luciferase activity in each sample normalized to represent RLU/mg of extracted protein. The procedure was approved by the University of Science and Technology of China Animal Care and Use Committee. In Vivo Distribution of Polyplexes. Dispersions of the Cy-5 DNA/cationic polymer polyplex were injected into mice through the tail vein. The mice were sacrificed 48 h after injection. The lung, liver, spleen, heart, and kidneys were excised and subjected to fluorescent image analysis using a Xenogen IVIS Lumina system (Caliper Life Sciences, Alameda, CA). The procedure was approved by the University of Science and Technology of China Animal Care and Use Committee.

EXPERIMENTAL SECTION

Synthesis of PEG-Based Branched Polymer. 2-(2Methoxyethoxy)ethyl methacrylate (MEO2MA, mo1ecular weight is 188 Da, 1.316 g, 7 mmol), oligo(ethylene glycol) methacrylate (OEGMA, molecular weight is 475 Da, 1.425 g, 3 mmol), N,N′cystaminebis(acrylamide) (CBA, 0.520 g, 2 mmol), cumyldithiobenzoate (CBD, 0.0298 g. 0.1 09 mmol), and azobis(isobutyronitrile) (0.0078 g, 0.047 mmol) were added in a polymerization tube with THF (18.0 mL). The reaction mixture was vacuum-sealed after degassing by three freeze−pump−thaw cycles and placed in a 60 °C oil bath for 15 h. Subsequently, a PEG-based branched polymer was obtained by precipitating into an excess of hexane, and dried under vacuum. Mn is 48 700 and PDI is 1.9. The molar percentages of MEO2MA, OEGMA, and CBA in the polymer are 58.0, 28.1, and 13.9%, respectively. Its LCST is 45 °C. Animals. Male ICR mice (Vital River Laboratories, Beijing, China) were used at the age of 7 to 8 weeks and received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedure was approved by the University of Science and Technology of China Animal Care and Use Committee. Three mice per sample were used for each condition. Hemolysis Test. Fresh blood was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Erythrocytes were washed three times by centrifugation at 1500 rpm. A total of 200 uL of the whole blood was added to different amounts of polyplex solutions, and the volume was adjusted to 1 mL with PBS (pH 7.4). The hemolysis test in PBS solution and in Triton X-100 solution (1%, v/v) was used as negative and positive controls, respectively. After incubation at 37 °C for 3 h, the solutions were centrifuged at 2000 rpm for 10 min. A total of 200 uL of the supernatant was collected and seeded in each well of 96-well plate. The absorbance was recorded at a wavelength of 540 nm. The percentage hemolysis was calculated using the following equation

percentage of hemolysis = (As − An)/(Ap − An) × 100% where As, An, and Ap are the absorbance of sample, negative, and positive controls, respectively. Protein BSA Adsorption Assay. BSA solution (1.0 mL, 2.0 mg/ mL) was mixed with 1.0 mL of PEI/DNA polyplex and PEI/DNA@ PEG(4), PEI/DNA@PEG(10), PEI/DNA@PEG(20), and shaken at 37 °C for 30 min. The mixtures were centrifuged, and the supernatants were collected. The BSA concentration in the supernatant was determined using a bicinchoninic acid (BCA) protein assay using a calibration curve constructed with standard bovine serum albumin solutions (Pierce). Stability of the Polyplex Nanoparticles in the Presence of NaCl. The polyplex solution was adjusted to 20 μg/mL of DNA and 150 mM NaCl by adding NaCl solution. Ethidium bromide (EtBr) was mixed with the solution, which was diluted to a final concentration of 2.5 μg/mL. After incubating for 30 min, 100 μL of the polyplex-EtBr solution was added to each well of a 96-well microplate. The fluorescence of the solution was measured for three times using a fluorescence microplate reader (Thermo Scientific Multiskan Flash, San Jose, CA) with λex = 510 nm, λem = 610 nm, and a 5 nm slit width. The fluorescence of the DNA/EtBr solution was set to 100%, and the background fluorescence was set to 0% using EtBr solution (2.5 μg/mL) alone. Agarose Gel Electrophoresis Assay. Polyplexes were loaded onto a 0.8% agarose gel and subjected to electrophoresis for 50 min at 90 V in TAE running buffer. The gel was soaked in 0.5 μg/mL EtBr for 20 min, then visualized under UV illumination on a Kodak Gel Logic 100 Imaging System. Coating Polyplex with Bioreducible PEG Nanoshell. First, DNA and PBS were combined to make the final concentration as 0.5 μg/ well to prepare the polyplex with a bioreducible nanoshell. Then, PEI was added to a final volume of 150 μL/well at N:P of 10. After incubation for 30 min, PEG-branched polymer was added to the well to tune the PEG branched polymer concentration to 1, 2, 4, 10, and 20 times the DNA concentration (defined, respectively, as PEI/DNA@ PEG(1), PEI/DNA@PEG(2), PEI/DNA@PEG(4), PEI/DNA@

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RESULTS AND DISCUSSION Preparation of Self-Cross-Linking PEG-Branched Polymer. Self-cross-linking PEG-based branched polymer was prepared via reversible addition−fragmentation chain transfer (RAFT) polymerization of oligo(ethylene glycol) methacrylate (OEGMA) and 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) using N,N′-cystaminebis(acrylamide) (CBA) as the branching unit.20,21 The prepared polymer is temperatureresponsive, and its lower critical solution temperature (LCST) is 45 °C (Supplementary Figure S2 in the Supporting Information). The disulfide-containing PEG-branched polymers will chemically self-cross-link above 45 °C via intermolecular disulfide exchange.20,22−25 The storage modulus G′ and loss modulus G″ curves as a function of time or frequency (ω) are a good experimental estimate of the gel transition (cross-linking). The values for G′ and G″ of the prepared PEG-based branched polymer slightly increase with time at 25 °C, and the G′ and G″ curves do not intersect, indicating that this PEG-based branched polymer will not cross-link at 25 °C. However, at 50 °C, the values for G′ and G″ of the PEG-based branched polymer also increase with time and the G′ and G″ do intersect, indicating that PEG-based branched polymer can chemically self-cross-link via 2908

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Figure 1. (a) Illustration depicting the growth of a bioreducible PEG-based nanoshell onto polyplexes via heating the polyplex dispersions in the presence of PEG-based branched polymers and GSH triggering the opening of this nanoshell. (b) Zeta potential distribution of the PEI/DNA polyplex, PEI/DNA@PEG(10), and PEI/DNA@PEG(10) after being treated with GSH. (c) Hydrodynamic diameter, measured via dynamic light scattering, of the polyplex, PEI/DNA@PEG(10), and PEI/DNA@PEG(10) after being treated with GSH.

upon heating. The chemically cross-linking occurs via intermolecular disulfide exchange between the disulfide bonds in the backbone of PEG-based branched polymer20,22−25 upon heating, cross-linking the polymer (Figure S3 in the Supporting Information). Growing a Bioreducible Nanoshell onto the PEI Polyplex. Figure 1a illustrates the growth of a bioreducible

nanoshell onto polyplex via slightly heating the polyplex dispersions in the presence of PEG-based branched polymers. A polyplex of branched PEI (MW = 25 k) and pDNA was first prepared, with a size of 177 nm and N/P ratio of 10. Subsequently, the PEG-based branched polymer (the weight ratio of PEG/DNA is 10) was added to the polyplex solution, and the system was slightly heated to 50 °C. Under 50 °C, PEG-based 2909

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branched polymers liberate absorbed water molecules and its solubility become poor, and hence they shrink and collapse together into small nanoparticles. The formed polymer nanoparticles are not stable in water, and they are absorbed onto the surface of PEI/DNA polyplex nanoparticles (Figure 1a).25 This procedure is similar to the growth of a poly(N-isopropylacrylamide) shell onto silica nanoparticles via seed precipitation polymerization.26−29 These absorbed nanoparticles are very soft, and they become closely packed around the polyplex “seed”, whereupon intermolecular disulfide-exchanges occur to crosslink the PEG aggregates at 50 °C and form the bioreducible nanoshell around the polyplex.22−25 The PEI/DNA polyplex with a PEG-based nanoshell is identified as PEI/DNA@ PEG(10) at a PEG/DNA weight ratio of 10. The zeta potential of the PEI polyplex is 25.0 mV, but this value decreases to 0.63 from 25.0 mV after slightly heating the polyplex dispersions in the presence of PEG-based branched polymers for 30 min (Figure 1b). This indicates that a PEG-based nanoshell has been grown on the surface of the PEI/DNA polyplex. Dynamic light scattering (DLS) shows that PEI/DNA polyplex has a hydrodynamic diameter of 177 nm, which increases to 211 nm after slightly heating the polyplex dispersions in the presence of PEGbased branched polymers for 30 min.(Figure 1c). All of these results indicate that the PEG-based nanoshell has grown around the PEI/DNA polyplex. Furthermore, after PEI/DNA@ PEG(10) is treated with DTT, its size decreases to 184 nm (Figure 1c), which is very close to the original size of the PEI polyplex, indicating that the nanoshell on PEI/DNA polyplex can be opened by DTT. The zeta potential results show that PEG can completely cover the PEI/DNA polyplex when the weight ratio of PEG to DNA is greater than 10, at a N/P ratio of 10. PEG cannot completely cover PEI/DNA polyplex if the weight ratio of PEG to DNA is below 10 (Figure S5 in the Supporting Information). Cytotoxicity of PEI Polyplex with Bioreducible Nanoshell. Many cationic vectors for gene therapy exhibit substantial toxicity, which has limited their clinical applicability.9 The main reason is that the excess positive charges on the surface of the complexes can interact with cell membranes7 and inhibit normal cellular processes. Such processes could include clathrinmediated endocytosis,30 the activity of ion channels, of enzymes and membrane receptors,7 and cell-survival signaling, inducing cell necrosis, apoptosis, and autophagy.31 Covering a bioreducible PEG-nanoshell onto PEI/DNA polyplex changes the positively charged surface to a neutral surface, which can reduce the cytotoxicity. The cytotoxicity of PEI/DNA@PEG and PEI/ DNA polyplex is evaluated in HeLa cell lines by MTT assays. As shown in Figure 2a, PEI/DNA@PEG exhibits much lower cytotoxicity compared with PEI/DNA polyplexes. This result agrees well with the accepted opinion that the cytotoxicity is strongly correlated with the surface charge density of the polyplex.32−34 Protein BSA Adsorption. During gene delivery, many polyplexes can absorb proteins. Proteins adsorbed onto the surface of a polyplex can trigger thrombosis and blood coagulation, leading to life-threatening situations. BSA adsorption of PEI/DNA polyplex is much higher than that of PEI/ DNA@PEG, as shown in Figure 2b. The absorption is induced mainly by electrostatic interactions between negatively charged BSA and the positively charged polymer.34 The protein adsorption value for PEI/DNA@PEG(10) is about one-third of that for the PEI/DNA polyplex.

Figure 2. (a) Cell viability for the PEI/DNA polyplex, PEI-g-PEG/DNA complex and PEI/DNA@PEG(4), PEI/DNA@PEG(10), and PEI/ DNA@PEG(20). (b) Protein adsorption for the PEI/DNA polyplex, PEI-g-PEG/DNA complex, PEI/DNA@PEG(4), PEI/DNA@ PEG(10), and PEI/DNA@PEG(20). (c) Hemolysis test results for the PEI/DNA polyplex, PEI-g-PEG/DNA complex, PEI/DNA@ PEG(4), PEI/DNA@PEG(10), and PEI/DNA@PEG(20) at PEI concentrations of 300, 500, or 100 μg/mL (N/P = 10).

Hemolysis Test. The compatibility of a gene delivery vector with blood indicates its suitability for introduction into systemic circulation. It has been reported that PEI can destabilize the plasma membrane of red blood cells, inducing cell necrosis, apoptosis, and autophagy.9,34 As shown in Figure 2c, PEI/ DNA@PEG(10) induces much less hemolysis than PEI/DNA and PEI-g-PEG/DNA. (Each PEI chain has been grafted with ∼13 PEG chains, prepared via branched PEI with molecular weight of 25 kDa reacting with PEG with molecular weight of 480 (see Figure S4 in the Supporting Information).) At PEI concentration of 300 and 500 μg/mL, PEI/DNA complex induces 31 and 47% hemolysis, respectively. In contrast, PEI/ DNA@PEG(10) induces only 4.3% hemolysis at 300 μg/mL and 7.2% hemolysis at 500 μg/mL. The high hemolytic damage rate of PEI may result from its high charge density, which can undergo excessively strong interactions with membrane proteins and 2910

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electrostatic interactions between polymer and DNA are inhibited by the PEG moiety, leading to weaker binding that may result in less compact structures, decreasing the cellular uptake. The low cellular uptake of PEI/DNA@PEG(1) and PEI/ DNA@PEG(4) may result from the incomplete PEG coverage of the polyplex in these systems. The low cellular uptake of PEI/ DNA@PEG(20) may result from the large size (∼350 nm) of PEI/DNA@PEG(20). PEI/DNA@PEG(10) is stable in the presence of serum, showing the highest cellular uptake. Gene Transfection in Vitro. Because the polyplex with bioreducible nanoshell shows low cytotoxicity, high cellular uptake, high stability, and good compatibility with blood, it should have much high gene transfection efficacy in vivo compared with the bare polyplex. The transfection efficiency of the polyplex with bioreducible nanoshell, in the presence of 10 and 50% serum, was studied (Figure 4b,c) in HEK-293T cells. In the control experiment, it is clear that the transfection efficiency of PEI/DNA polyplex is only slightly increased when PEG is added (as shown in Figure 4b). In the presence of 10%, serum, the PEI/DNA polyplex, and PEI-g-PEG/DNA polyplex show low transfection activity compared with PEI/DNA@PEG(10). The low transfection activity of the PEI/DNA polyplex and PEIg-PEG/DNA polyplex may result from the serum causing the polyplexes to dissociate before they have entered the cell. In the presence of 50% serum, PEI/DNA@PEG(10) shows the highest transfection activity compared with the PEI-g-PEG/DNA and PEI/DNA polyplexes. Gene Transfection in Vivo. PEI/DNA polyplex and PEI/ DNA@PEG(10) were injected into animals through the tail vein (20 μg total DNA in 200 μL/mouse). Organs including heart, lung, liver, spleen, and kidney were dissected after the injection for 48 h. In Figure 4d, the luciferase activity in each organ is normalized to represent RLU/mg of extracted protein; the results show that PEI/DNA@PEG(10) has much higher activity than the PEI/DNA polyplex in the lung, liver, spleen, heart, and kidney. We also explored the biodistribution of Cy5-DNA in mice via intravenous injection with PEI/Cy5-DNA polyplex, PEI-g-PEG/Cy5-DNA polyplex, and PEI/Cy5-DNA@ PEG(10). The organs were collected for fluorescence imaging using a Xenogen IVIS system. As is shown in Figure 4e, significantly enhanced fluorescence is observed using PEI/Cy5DNA@PEG(10). All of these indicate that the polyplex with a bioreducible nanoshell should have much high gene transfection efficacy in vivo. Disulfide-containing branched PEG polymer prepared via RAFT polymerization using N,N′-cystaminebis(acrylamide) (CBA) as the branching unit produces branched polymer with some vinyl terminal units (as shown in Scheme S1 and Figure S1 in the Supporting Information), which makes it very easy to link RGD, mannose, and folate onto the surface of the nanoshell via Michael addition of amines to RGD, mannose, and folate with vinyl terminals at 37 °C. The gene transfection efficiency of PEI/ DNA@PEG(10) with RGD, mannose, and folate is higher than that of PEI/DNA@PEG(10) (Figure S6 in the Supporting Information). Taken together, all of these results show that growing a bioreducible PEG nanoshell onto a polycation/DNA polyplex is an ideal method to protect the polyplex and improve its performance as a vector for gene therapy.

phospholipids and thus disturbs membrane structure and function.35 The PEG moieties on PEI/DNA@PEG(10) can help to avoid the disruption of blood cell membranes. Stability in the Presence of Salt or Heparin. The vectors should pass through physiological fluid containing serum components and salts, in which polyplex vector tend to break apart or aggregate, to deliver the genes to target cells.8,9 The stability of PEI/DNA@PEG(10) in the presence of heparin is evaluated by the agarose gel electrophoresis. As shown in Figure 3a, in the presence of heparin, bands corresponding to free DNA

Figure 3. (a) Agarose gel electrophoresis results of PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10) with or without heparin (HP) at a HP concentration of 50 μg/mL). (b) Change in the fluorescence intensity of the PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10) before and after adding NaCl.

are clearly observed for the PEI/DNA complex, PEI-g-PEG/ DNA complex, and PEI/DNA+PEG system, while almost no free DNA is detected for PEI/DNA@PEG(10). This result indicates the substantial stability of PEI/DNA@PEG(10) in the presence of heparin, where the PEI/DNA polyplex and PEI-gPEG/DNA polyplex are unstable. In the presence of ethidium bromide, the addition of NaCl into the PEI/DNA, PEI-g-PEG/ DNA, PEI/DNA+PEG and PEI/DNA@PEG(10) PEI/DNA, PEI-g-PEG/DNA, and PEI/DNA+PEG systems substantially increases the fluorescence (Figure 3b), whereas the change of fluorescence intensity observed for PEI/DNA@PEG(10) system is very little (Figure 3b). This result suggests that the PEG surrounding the PEI/DNA@PEG(10) system inhibits the ability of NaCl to bind the polycation in the polyplex and inhibits the regeneration of ethidium bromide binding sites in the DNA. The polyplex with a bioreducible PEG-based nanoshell is thus stable in the presence of NaCl, while the PEI/DNA polyplex and PEI-gPEG/DNA polyplex are unstable in the presence of NaCl. Cellular Uptake. Cellular uptake plays an important role in successful gene transfection. The cellular uptake results of PEI/ DNA, PEI-g-PEG/DNA, and PEI/DNA@PEG in the presence of 10% FBS are shown in Figure 4a. It is obvious that PEI/ DNA@PEG(10) has the highest cellular uptake. This is because the PEI/DNA polyplex alone tends to aggregate in the presence of serum. PEI-g-PEG/DNA tends to break apart because the

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CONCLUSIONS A new and facile method for growing a bioreducible nanoshell onto a polycation/DNA polyplex is reported, in which slight heating induces the shrinking and collapsing of a PEG-based 2911

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Figure 4. (a) Cellular uptake of PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG at PEG/DNA weight ratios of 1, 4, 10, and 20. (b) Gene transfection efficiency of PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10) in HEK-293T cell in the presence 10% serum. (c) Gene transfection efficiency of PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10) in HEK-293T cell in the presence 50% serum. (d) Transfection efficiency of PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10) in different organs. (e) Fluorescence images of organs after injecting with PEI/DNA polyplex, PEI-g-PEG/DNA complex, and PEI/DNA@PEG(10).

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hyperbranched polymer onto the polyplex. Subsequent intermolecular disulfide exchange occurs to cross-link the PEG aggregations, forming the bioreducible nanoshell. The nanoshell isolates the polyplex from serum components and salts in physiological fluids. When inside the cell, the nanoshell opens upon reduction by GSH, to release the polyplex. The polyplex with a bioreducible nanoshell shows high gene transfection, low hemolysis, low protein absorption, and low cytotoxicity. The simplicity of this method provides a promising platform for improving the performance of polycation/DNA polyplexes as gene vectors.

ASSOCIATED CONTENT

S Supporting Information *

Detailed polymer synthesis procedure and characterization. This material is available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2912

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(29) Yan, L.; Wang, Z. K.; Yan, J. J.; Han, L. F.; Zhou, Q. H.; You, Y. Z. Polym. Chem. 2013, 4, 1243. (30) Gao, X. L.; Yao, L.; Song, Q. X.; Zhu, L.; Xia, Z.; Xia, H. M.; Jiang, X. G.; Chen, J.; Chen, H. Z. Biomaterials 2011, 32, 8613. (31) Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. J. Controlled Release 2006, 114, 100. (32) Fischer, D.; Bieber, T.; Li, Y. X.; Elsasser, H. P.; Kissel, T. Pharm. Res. 1999, 16, 1273. (33) Kircheis, R.; Wightman, L.; Wagner, E. Adv. Drug Delivery Rev. 2001, 53, 341. (34) Sun, J.; Zeng, F.; Jian, H. L.; Wu, S. Z. Biomacromolecules 2013, 14, 728. (35) Luo, X. H.; Huang, F. W.; Qin, S. Y.; Wang, H. F.; Feng, J.; Zhang, X. Z.; Zhuo, R. X. Biomaterials 2011, 32, 9925.

ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (grant numbers 51033005, 21074121, 21090354, 51273187, and 81171829), the Fundamental Research Funds for the Central Universities (WK2060200012), and the Program for New Century Excellent Talents in Universities ((NCET-11-0882)

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dx.doi.org/10.1021/bm500518u | Biomacromolecules 2014, 15, 2907−2913