Synergistic Effect of PEI and PDMAEMA on Transgene Expression in

May 18, 2015 - DNA polyplexes at a ratio of 1:3 or 1:9 (PDMAEMA: PEI), depending on cell type, in ... from endosomes, facilitating their later express...
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Synergistic Effect of PEI and PDMAEMA on Transgene Expression in Vitro Chia-Wen Lo,†,# Wei-Hao Liao,†,# Chueh-Hung Wu,†,‡ Jyun-Lin Lee,† Ming-Kuan Sun,⊥ Hui-Shan Yang,∥ Wei-Bor Tsai,§ Yung Chang,*,∥ and Wen-Shiang Chen*,†,⊥ †

Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan ROC ‡ Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan ROC § Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan ROC ∥ R&D Center for Membrane Technology and Department of Chemical Engineering, Chung Yuan Christian University, Jhong-Li, Taoyuan, Taiwan ROC ⊥ Division of Medical Engineering Research, National Health Research Institutes, Miaoli, Taiwan ABSTRACT: Polyethylenimine (PEI) and poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) have both been used for DNA delivery. PDMAEMA has been shown to exhibit better gene transfection efficiency but lower expression ability than PEI. We mixed the two polymers at different ratios to investigate whether the resulting “dual” polyplex (PEI/ PDMAEMA/DNA) could enhance both gene transfection efficiency and DNA expression ability. Experimental results showed a significant increase in DNA internalization and DNA expression for the PDMAEMA/PEI/ DNA polyplexes at a ratio of 1:3 or 1:9 (PDMAEMA: PEI), depending on cell type, in comparison with PEI/DNA, PDMAEMA/DNA, and PDMAEMA/PEI/DNA at other ratios. PDMAEMA/PEI/DNA polyplexes did not reduce cell viability. In contrast to with the conventional approach using covalently modified PEI, the proposed “combination” approach provided a more convenient and effective way to improve transgene expression efficiency.

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to deliver larger DNA payloads.8 In addition, polyplexes under optimal conditions may also prolong the expression of therapeutic genes and improve their therapeutic effects.9 The most commonly used polymer for DNA delivery is polyethylenimine (PEI), which is commercially available in linear (as LPEI, 22 kDa) and branched (as BPEI, 25 kDa) forms. Both forms are extensively used in gene delivery for their high levels of gene expression. PEI has high density positivecharged amine groups, thus forming nanoparticle complexes (polyplexes) by binding to negatively charged DNA molecules. Through endocytosis, PEI interacts with negatively charged cell membranes and internalizes itself into the cell. After crossing the membranes, PEI induces osmotic swelling and forces the release of PEI/DNA polyplexes or dissociated free-form DNA from endosomes, facilitating their later expression.10 PEI has also been shown to protect combined DNA from enzyme digestion.11

INTRODUCTION Gene therapy has been shown to have great potential to treat certain diseases such as cancers, cardiovascular diseases, and inherited immune deficiencies, which could not be treated with conventional drug and protein therapeutic regimens. Because of ease of use and high efficiency, viral vectors have been predominantly used for gene therapy; however, clinical applications of viral vectors were limited due to concerns about their safety and immunogenicity.1,2 Therefore, nonviral vectors for gene internalization have been reported to be safer and easier to use, such as electroporation, cationic polymer, liposomes, gene guns, hydrodynamics, and ultrasound.3 Among them, polymer-mediated gene transfer has received considerable attention recently.4,5 Through electrostatic interactions, cationic polymers interact with negatively charged DNA and spontaneously bind and condense to form nanoparticles. The total charge of polyplexes maintains a positive net value, enabling carriers to efficiently interact with the negatively charged cell membranes to facilitate the internalization of DNA into cells through endocytosis pathways.6,7 Advantages of cationic polymers include ease of use, stability, low immunogenicity and toxicity, and the ability © 2015 American Chemical Society

Received: February 9, 2015 Revised: May 1, 2015 Published: May 18, 2015 6130

DOI: 10.1021/acs.langmuir.5b00520 Langmuir 2015, 31, 6130−6136

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Langmuir Table 1Concentration of Polymer for Each Group concentration groups

PDMAEMA

PEI

1:9

1:3

1:1

3:1

9:1

16.0 0

0 4.33

1.6 3.87

4.0 3.23

8.0 2.16

12.0 1.07

14.4 0.43

PDMAEMA (μg/mL) PEI (μg/mL)

Culture Collection (Manassas, VA). These cells were cultured in Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM, High Glucose, Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum (Gibco), and a 1% mixture of penicillin G, streptomycin, and amphotericin B (Gibco) at 37 °C in 5% CO2. Measurement of Zeta-Potential and Particle Size. The spontaneous electrostatic formation of stable nanoparticle complexes was induced by mixing cationic PEI, PDMAEMA, or PEI/PDMAEMA with negatively charged DNA. Two micrograms of DNA was mixed with the PEI, PDMAEMA, or different mixing ratios of PEI/ PDMAEMA at the fixed NP ratio of 10. The formed polyplexes were diluted in 150 mM NaCl. Particle sizes and zeta potentials of the PDMAEMA/DNA, PEI/DNA, or PEI/PDMAEMA/DNA polyplexes were evaluated using the Nano-ZS (Malvern, Worcestershire, U.K.). The presented data are the means of at least three measurements. To evaluate the binding conditions of plasmid DNA (2 μg), we incubated a mixture of PEI, PDMAEMA, or PEI/PDMAEMA of various ratios and plasmid DNA at 37 °C for 10 min. The mixture was centrifuged at 12 000g and the supernatant (nonbinding DNA) was collected and quantified by measuring its absorbance at 260 nm. The binding percentage is defined as (total amount of DNA − amount of nonbinding DNA)/total amount of DNA. Stability Assay of PDMAEMA/DNA, PEI/DNA, or PEI/PDMAEMA/ DNA Polyplexes. To compare the protection ability of PDMAEMA and PEI/PDMAEMA against deoxyribonuclease (DNase) degradation with PEI, we incubated tested polyplexes for 30 min at 37 °C in the DNase I reaction buffer (100 mM Tris−HCl, PH 7.5 and 25 mM MgCl2) containing 0.5 unit/μL of DNase I. The solution was incubated with 1 μL of EDTA and heated to 70 °C for 10 min to deactivate DNase I prior to electrophoresis. Each sample was treated with SDS and incubated at 37 °C for 60 min to dissociate DNA from complexes. The DNA was then electrophoresed on a 1% agarose gel and stained with SYBR Safe DNA gel stain (Invitrogen, Cergy Pontoise, France). Gene Delivery Evaluation. PEI is well known to facilitate both in vitro and in vivo gene transfection.16,17 Thus, the transfection ability of PDMAEMA/DNA and PEI/PDMAEMA/DNA polyplexes of various PEI and PDMAEMA mixing ratios was compared with that of the PEI/DNA polyplexes in BNL and NIH3T3 cells. Experiments were performed at a constant DNA (2 μg) mixed with PDMAEMA, PEI, or PEI/PDMAEMA at N/P ratio of 10. BNL cells and NIH3T3 cells were seeded in 24-well plates at an initial density of 5 × 104 in 1 mL of growth medium and incubated for 24 h. The culture medium contained 2 μg of plasmid DNA with a predetermined PDMAEMA, PEI, or PEI/PDMAEMA mixture. One day after treatment, the cells were stripped from the culture plates and suspended in a luciferase lysis buffer (CCLR, Promega, Madison, WI). The cell suspensions were mixed and then centrifuged at 12 000g at 4 °C for 10 min. The supernatant was assayed with a luciferase assay substrate kit (Luciferase Assay System, Promega, Madison, WI). Luciferase activity was measured by a microplate luminometer (Infinite M200, Tecan, Austria) and was normalized to the protein content of the cells. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Cell Viability Assay. In vitro cytotoxicity tests were performed using MTT assay. Cells were seeded in 24-well plates at an initial density of 5 × 104 in 1 mL of a growth medium and incubated overnight. For each well, we added 2 μg of DNA into 50 μL of 150 mM NaCl and added 0.23−9.6 μg of different polymers into 50 μL of 150 mM NaCl and then mixed the 50 μL polymer solution with the 50 μL DNA solution. The solution was mixed immediately and incubated for 10

Another polymer, poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA), has also been shown to be a potential polycationic carrier to facilitate gene and transfection efficiency, both in vitro and in vivo.12−15 Unlike PEI, PDMAEMA could be synthesized from its monomer, 2-(dimethylamino) ethyl methacrylate or DMAEMA, to produce linear polymers with similarly repetitive units or complex branched structures with higher homogeneity in a well-controlled living polymerization. Unlike LPEI (secondary amine) and BPEI (mixed primary, secondary and tertiary amines), PDMAEMA is a pure tertiary amine structure. A prior study showed that, similar to linear PEI, PDMAEMA could facilitate gene transfection and prolong the expression duration both in vitro and in vivo.12 PDMAEMA provides better DNA internalization and less toxicity than PEI does in vitro.15 PDMAEMA/DNA at low weight ratios (0.25 and 0.5) provides good transfection efficiency for in vivo systems; however, the overall in vitro expression ability of the transfected DNA by PDMAEMA polyplex is lower than that of the PEI/DNA. The exact mechanism that PDMAEMA/DNA has high DNA internalization but low expression ability is unclear. OCombining PDMAEMA and PEI may preserve their advantages while minimizing their disadvantages. In this study, we aim to (1) test the hypothesis that mixing PDMAEMA and PEI to form a “dual” polyplex (PEI/ PDMAEMA/DNA) could enhance not only DNA internalization ability but also the overall expression of transfected DNA and (2) propose a mechanism by which PDMAEMA/ DNA exhibits higher DNA internalization but lower expression ability than PEI/DNA.

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EXPERIMENTAL SECTION

Preparation of PDMAEMA Polycation and Branched PEI. PDMAEMA polycation was prepared as described in a previous study.9 The polymer was dried in a freeze-dryer at −45 °C to yield a white powder with a molecular weight of 6 kDa (polydispersity index = 2.755) and 39 repeat units. Molecular weight distributions of the prepared PDMAEMA polycation were determined by aqueous gelpermeation chromatography (GPC). Aqueous GPC was performed using two Viscogel columns, a G4000 PWXL and a G6000 PWXL (the range of molecular weight was from 2 kDa to 8000 kDa), connected to a model Viscotec refractive-index detector at a flow rate of 1.0 mL/min and a column temperature of 23 °C. The eluent was an aqueous solution composed of 0.1 M NaNO3 at pH 7.4. Poly(ethylene oxide) (PEO) standards from Polymer Standard Service (Warwick, RI) were used for calibration. Branched PEI (BPEI; Mw = 25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO). Expression Vector and Polyplex Preparation. The pCI-neo-luc was constructed by subcloning firefly luciferase cDNA (luc) into the pCIneo Vector. Competent Escherichia coli DH5α was used for plasmid pCI-neo-luc transformation, and endotoxin-free plasmid DNA was purified using the Qiagen EndoFree Plasmid Max kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. PDMAEMA/DNA, PEI/DNA, or PEI/PDMAEMA/DNA polyplexes of various predetermined PEI to PDMAEMA ratios (nitrogen ratios 1:9, 1:3, 1:1, 3:1, and 9:1) were prepared with an N/P ratio of 10 (i.e., ratio of overall nitrogen atoms on PDMAEMA, PEI, or PEI/ PDMAEMA mixtures to phosphates on DNA). Cell Culture. BNL 1MEA.7R.1 (chemically transformed liver cells) and NIH 3T3 fibroblast cells were purchased from American Type 6131

DOI: 10.1021/acs.langmuir.5b00520 Langmuir 2015, 31, 6130−6136

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Langmuir min at room temperature. Table 1 summarizes the concentration of polymer for each group. After 24 h of treatment with PEI/DNA, PDMAEMA/DNA, or PEI/ PDMAEMA/DNA polyplexes, the cells were treated with MTT reagent (Sigma-Aldrich, St. Louis, MO) and were further incubated for 4 h at 37 °C. The medium was removed and 1 mL of DMSO was added to dissolve the MTT product (formazan crystals). Complete dissolution was achieved by gently shaking the plate for 15 min. Aliquots (100 μL) of the resulting solution were transferred to 96-well plates, and the microplate spectrophotometer system (Infinite M200, Tecan, Austria) was used for recording absorbance at 570 nm. Relative cell viability was calculated as (Atreat/Acontrol) × 100%. Quantification of DNA Uptake. The amount of DNA transfected into cultured cells was quantified by flow cytometry 24 h following the initiation of the transfection process to evaluate the ability of PDMAEMA/DNA, PEI/DNA, or PEI/PDMAEMA/DNA polyplexes in uptake of plasmid DNA. pCI-neo-Luc DNA was labeled using a Fluorescein Label IT Tracker Kit (Mirus Bio Corporation, Madison, WI) for measurements of DNA uptake. Subsequently, fluoresceinlabeled DNA was used to prepare for PEI, PDMAEMA and different mixing ratios of PEI/PDMAEMA polyplexes at an N/P ratio of 10. The fluorescein-labeled PDMAEMA/DNA, PEI/DNA, or PEI/ PDMAEMA/DNA polyplexes were added to cultured BNL cells (5 × 104 cells/1 mL) and incubated for 24 h in 24-well plates. Intracellular uptake of DNA was measured by a LSRII flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Treestar Software, San Carlos, CA). Statistical Analysis. All data are presented as the mean ± SEM. The differences were analyzed using Student’s t test or one-way ANOVA. “P” 90%) contained fluoresceinlabeled plasmid DNA 24 h after transfection. As for the internalized fluorescein-labeled plasmid DNA, the fluorescence intensity in the PDMAEMA, DMAEMA/PEI (1:3), and PDMAEMA/PEI (1:9) groups was larger than that in the PEI group.

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RESULTS Physicochemical Characterization of PDMAEMA/DNA, PEI/DNA, and PEI/PDMAEMA/DNA Polyplexes. Table 2 Table 2. Particle Sizes and Zeta Potentials of Plasmid DNA (2 μg) Mixed with Different Mixing Ratios of PEI/ PDMAEMA at NP ratio of 10a N/P ratios: 10 (2 μg) PDMAEMA PEI PDMAEMA:PEI PDMAEMA:PEI PDMAEMA:PEI PDMAEMA:PEI PDMAEMA:PEI

1:9 1:3 1:1 3:1 9:1

particle size (nm) 146.6 518.6 154.0 218.0 261.7 230.0 160.7

± ± ± ± ± ± ±

3.4 53.1 5.7 10.6 12.7 6.8 4.3

zeta potential (mV) 23.2 21.9 28.6 26.4 24.3 23.7 24.9

± ± ± ± ± ± ±

0.8 0.2 0.3 0.2 1.5 0.5 0.2

Values are mean ± SEM for three to four independent measurements.

a

shows the average sizes and zeta potentials for polyplexes formed at a constant DNA concentration of 2 μg and an N/P ratio of 10. The mean particle size is significantly larger for the PEI group but not significantly different among PDMAEMA and PEI/PDMAEMA groups of various mixing ratios (nitrogen ratios). All groups are positively charged. As shown in Figure 1, almost 100% of the DNA was captured by the PDMAEMA, PEI, or various PEI/PDMAEMA mixtures at an N/P ratio of 10, and thus no free form plasmid DNA remained in the solution. Protection of DNA against Degradation by Polymers. As shown in Figure 2, the naked DNA without polymer protection was quickly degraded by DNase I. The bright double bands in the PEI+DNase I column suggest that PEI exhibits a better protection effect against DNase I degradation than does PDMAEMA. Moreover, when polyplexes were formed by

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DISCUSSION In this study, we found that mixing PDMAEMA and PEI can significantly increase gene transfection level and intracellular DNA uptake without significant cytotoxicity. There seemed to be an optimal ratio of PDMAEMA/PEI for enhancement of gene transfection. Simply mixing PDMAEMA and PEI enhanced gene transfection efficiency without sacrificing cell viability. Polymer-mediated gene transfection efficiency of is affected by many factors such as molecular weight, surface charge, 6132

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Figure 2. Electrophoresis assay of PDMAEMA, PEI or PEI/PDMAEMA protects DNA against degradation.

Figure 3. In vitro transgene expression with PDMAEMA/DNA, PEI/ DNA or PEI/PDMAEMA/DNA at an N/P ratio of 10 in BNL (A) and NIH 3T3 (B). * p < 0.05; ** p < 0.001 vs. PEI.

Figure 4. Cell viability was assessed by MTT assay 24 h after treatment with PDMAEMA/DNA, PEI/DNA, or PEI/PDMAEMA/ DNA at an N/P ratio of 10. C is the control group without the addition of DNA/polymer. Results are presented as the mean ± SEM of three independent experiments. * p < 0.05 v.s. C.

charge density, hydrophilicity and the structure of cationic polymers. In the past decade, PEI has been chemically modified to decrease the impact of side effects and improve transfection 6133

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Figure 5. Effects of PDMAEMA, PEI, or PEI/PDMAEMA on the intracellular uptake of DNA monitored by flow cytometry 24 h after transfection. Fluorescence-labeled DNA was mixed with PDMAEMA, PEI, or PEI/PDMAEMA in culture cell plates. The percentage of cells with fluorescence (A) and the overall fluorescence intensities of cultured cells (B) are also shown. Control is the group without addition of DNA/polymer. Confocal images of fluorescence labeled DNA (C) for BNL cells treated with DMAEMA (a), PEI (b), 1:9 (c), and 1:3 (d). Green: Fluorescein labeled DNA. The cells were transfected for 24 h. Results are presented as the mean ± SEM of three independent measurements. * p < 0.05 versus PEI group.

efficiency. For example, hydrophobic modifications of PEI can improve gene delivery efficiency,18 polysaccharide modification such as PEI-chitosan binding improves the biocompatible and biodegradable,19,20 PEGylation of PEI enhances the serum stability of polyplexes during circulation,21 and the attachment of recognizing molecules enhances targeting accuracy.22,23 Most PEI modifications use covalent linkages to decrease the impact of side effects and improve transfection efficiency. This study proposes a simple and convenient way to enhance PEI transfection efficiency by mixing with another well-studied polymer, PDMAEMA. We also found an optimal ratio of PDMAEMA/PEI (1:3 or 1:9, in different cell types) to provide better transfection efficiency than PEI or PDMAEMA alone. Mixing the two polymers rather than covalently combining them provides a way to streamline the manufacturing process, optimizing the advantages of the two polymers while minimizing their disadvantages.

Each polymer has its own advantages and disadvantages. A prior study showed that PDMAEMA provides better DNA internalization but low expression ability compared with those of PEI.9 The current study showed comparable results. Both the percentage of cells containing fluorescein-labeled plasmid DNA and the mean intracellular fluorescence intensity of the PDMAEMA group are significantly higher than those of the PEI group 24 h after transfection. However, the PDMAEMA/ DNA group was inferior to the PEI/DNA group as for the overall in vitro transfection efficiency. Therefore, PDMAEMA/ DNA polyplexes seemed to enable high intracellular uptake of plasmid DNA but their expression ability was limited. Two hypotheses for explaining the mismatch included (1) the tight bond between the PDMAEMA and DNA molecules which reduces the availability of free-form DNA capable of entering the nuclei for transcription, and (2) the fast degradation of freeform DNA by cytoplasmic DNase I due to under-protection of 6134

DOI: 10.1021/acs.langmuir.5b00520 Langmuir 2015, 31, 6130−6136

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PDMAEAM-m-PEI/DNA polyplex above that of the PEI/DNA polyplex. However, the component distribution in the nanoparticles may play a more important role for the enhanced gene transfection behavior. Because the main goal in the current study is to propose a more convenient and effective approach for enhancing transgene expression efficiency, further study should be conducted to disclose the exact mechanism that transgene expression efficiency can be improved simply by mixing two polymers. The current study is subject to one major limitation in that it reports in vitro results, as other studies have shown that in vivo conditions can differ significantly from in vitro conditions. Intra et al. reported that changing the N/P ratio of PEI/DNA affected the luciferase activity in certain in vitro conditions, but not in vivo.24 Our previous study also showed that optimal enhancement of transfection efficiency and expression duration occurred at different N/P ratios for in vitro (N/P ratio: 5) and in vivo (N/P ratio: 0.5) US-mediated gene transfection.25 The present study seeks an alternative, noncomplicate method of designing better nonviral vectors for gene therapy. In different conditions, such as different cell types and in vivo studies, it would be easier to test the optimal ratio of a mix than to design a new compound. For example, we have demonstrated the enhancement of transfection efficiency of two cell types at different mixing ratios, without sacrificing cell viability. Optimal mixing ratios for in vivo conditions would also exist, but further experiments are required.

DNA from DNase by PDMAEMA. Either overprotection or under-protection of the transfected DNA could result in the low expression ability of PDMAEMA/DNA polyplexes. Different from the PDMAEMA, PEI exhibits reduced internalization but better expression ability. As shown in Figure 2, PEI provides powerful protection for DNA against DNase I degradation, which was provided to a lesser degree by PDMAEMA. Almost no intact DNA was left after mixing DNase I with PDMAEMA/DNA polyplexes for 30 min. The PDMAEMA use in this study had a low molecular weight and the PEI used was in branch form. Therefore, when the ratio of PDMAEMA to PEI increased, the particle size became smaller. The branched form PEI may also provide a spatial barrier that preventing the DNA from the DNase, thus exerting better gene expression efficiency than PDMAEMA. Although a large amount of DNA was transfected into cells in the form of PDMAEMA/DNA polyplex by endocytosis, protection of DNA from DNase may not be as good as PEI. Mixing PEI with PDMAEMA overcame such disadvantages of PDMAEMA. We believe that the low gene expression ability of PDMAEMA/ DNA was more likely due to hypothesis 2: under-protection of the transfected DNA from the DNase by the PDMAEMA. Simply mixing PDMAEMA and PEI at a certain ratio was found to increase both the internalization and expression ability of plasmid DNA. Significant improvement to DNA internalization efficiency and DNA expression was found in the PDMAEMA/PEI/DNA groups at a ratio of 1:3 or 1:9, depending on cell types, without sacrificing cell viability. This improvement to transfection efficiency may result from the advantage of PDMAEMA in increasing the intracellular uptake of plasmid DNA and the advantage of PEI in protecting the internalized DNA against DNase I degradation. However, when the percentage of PDMAEMA increased in the mixture (PDMAEMA: PEI = 1:1, 3:1 and 9:1), the transgene expression dropped, probably due to reduced protection by PEI. Mixing PDMAEMA and PEI at a certain ratio was necessary to optimize the synergistic effects of the two polymers. Both PEI and PDMAEMA exhibited cytotoxicity at a certain level. In this study, we found that mixing PEI and PDMAEMA did not increase cytotoxicity in BNL cells. Furthermore, in NIH-3T3 cells, mixing PEI and PDMAEMA led to lower cytotoxicity than was achieved using PDMAEMA alone. For the polymerization of PDMAEMA, the increased molecular weight of the polymers is associated with the increased ratio of PDMAEMA monomer to APS initiator in the reaction solution. From the Viscotek GPC characterization and OmniSEC software analysis, the prepared PDMAEMA had an average molecular weight of 6 kDa and polydispersity of 1.5. Dynamic light scattering (DLS, Malvern Zetasizer Nano ZS) was used to characterize the hydrodynamic diameter and zeta potential of the prepared PDMAEMA, PEI and the mixed polymers (PDMAEMA-m-PEI) in a plasmid DNA solution with a controlled NP ratio of 10. The zeta potentials of PDMAEMA, PEI and all prepared PDMAEAM-m-PEI/DNA polyplex are in a positively charged range between 22 mV and 29 mV. As shown in Table 2, the different molar ratios of PDMAEMA and PEI affected the resulting size of the prepared PDMAEAM-m-PEI/DNA polyplex. The incorporation of PDMAEMA-rich composition in PDMAEMA-m-PEI mixed polymers was found to enhance the positive zeta potential when compared to individual homopolymers of PDMAEMA and PEI, and might be one of the reasons for the improved gene transfection efficiency and DNA expression ability of the

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CONCLUSIONS By mixing PDMAEMA and PEI, improved DNA internalization and transgene expression was achieved above that achieved by each individual polymer alone. Optimal mixing ratios were found for optimizing synergistic effects, which may result from increased intracellular uptake of plasmid DNA by PDMAEMA and the protection of the internalized DNA against DNase I degradation by PEI. An example is also provided for future studies on mixing different polymers with different advantages to achieve specific goals such as reduced toxicity, higher transfection efficiency, better expression, and long-lasting effect.

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AUTHOR INFORMATION

Corresponding Authors

*Y.C.: E-mail: [email protected]. Tel: +886-3-265-4122. *W.-S.C.: E-mail: [email protected]. Tel: +886-2-23123456, ext. 67087. Author Contributions #

C.-W.L. and W.-H.L. contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant MOST 103-2314-B-002011-MY3 from the Ministry of Science and Technology (MOST), ME-103-PP-03 from the National Health Research Institutes (NHRI), and 103-M2546 from the National Taiwan University Hospital (NTUH). We also thank the staff of the Biomedical Resource Core at the First Core Laboratories, National Taiwan University College of Medicine, for technical support. 6135

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(22) Kim, S. H.; Jeong, J. H.; Lee, S. H.; Kim, S. W.; Park, T. G. PEG conjugated VEGF siRNA for anti-angiogenic gene therapy. J. Controlled Release 2006, 116, 123−129. (23) Guo, W.; Lee, R. L. Receptor-targeted gene delivery via folateconjugated polyethylenimine. AAPS PharmSci 1999, 1, E19. (24) Intra, J.; Salem, A. K. Characterization of the transgene expression generated bybranched and linear polyethylenimine-plasmid DNA nanoparticles in vitro and after intraperitoneal injection in vivo. J. Controlled Release 2008, 130, 129−138. (25) Lee, J. L.; Lo, C. W.; Ka, S. M.; Chen, A.; Chen, W. S. Prolonging the expression duration of ultrasound-mediated gene transfection using PEI nanoparticles. J. Controlled Release 2012, 160, 64−71.

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