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Multilayered polyplexes with the endosomal buffering polycation in the core and the cell uptake-favorable polycation in the outer layer for enhanced gene delivery
Biomaterials 31 (2010) 9366e9372
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Multilayered polyplexes with the endosomal buffering polycation in the core and the cell uptake-favorable polycation in the outer layer for enhanced gene delivery Jin-He Ke a, Tai-Horng Young a, b, * a b
Institute of Polymer Science and Engineering, National Taiwan University, Taipei, 106, Taiwan, ROC Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2010 Accepted 18 August 2010 Available online 22 September 2010
In the present study, quaternary polyplexes were prepared by sequential addition of polycations (polyethylenimine (PEI) or poly (N-(8-aminooctyl)-acrylamide) (P8Am)) for loading pDNA into the core polyplexes and poly (acrylic acid) (PAA) for reversing charges to deposit additional polycation (PEI or P8Am) layer. It was found the cytotoxicity and cellular uptake expression of PEI core polyplexes could be improved by coating a cell uptake-favorable P8Am layer. Conversely, P8Am could not facilitate endosomal release through the proposed proton sponge effect so the PEI core was required for the P8Amcoated quaternary polyplexes to ensure efficient transfection. Consequently, an efficient and safe non-viral gene vehicle was constructed by layer-by-layer deposition, using alternate polyanion and polycation with required functionalities to overcome the obstacles met in the process of transfection. Maximum transfection activity with minimal toxicity was observed when the quaternary polyplex of pDNA/PEI/PAA/P8Am was prepared at a weight ratio of 1/1.5/3/5. Conversely, the same composition in different position such as the cell-favorable P8Am core was externally deposited with the endosome lytic moiety, PEI showed high toxicity and low efficiency. This indicates the pDNA/PEI/PAA/P8Am sequence for a quaternary polyplex is as important as the functional polymer selection for designing safe and reliable gene delivery vehicles. We demonstrate here that gene delivery efficiency may be improved by increasing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Gene therapy Gene vehicle Polyelectrolyte multilayer Polycation
1. Introduction Gene therapy offers a potential method to treat disease ranging from inherited disorders to acquired conditions and cancer by transferring exogenous nucleic acids into cells to alter protein expression profiles [1,2]. Generally, cationic nanoparticles would be appropriately applied to cellular uptake because of the electrostatic interaction between nanoparticles and cellular membrane, but it is limited by its low transfection efficiency and high cytotoxicity [3,4]. Except decreasing positive charge by chemical modification, “recharging” of polyplexes using polyanions to reverse the surface charge of polyplexes has been demonstrated to decrease the cytotoxicity of polycation/DNA complexes [5]. We have previously described the formation of ternary polyplexes containing pDNA, synthetic polycations and polyanions [6]. In this study, we extended
* Corresponding author. Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan. Tel.: þ886 2 23123456x81455; fax: þ886 2 23940049. E-mail address: [email protected] (T.-H. Young). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.08.066
these studies and found that the further addition of a polycationic layer on the ternary polyplexes could increase the levels of gene expression with reduced toxicity. Two cationic polymers, polyethylenimine (PEI) and poly (N-(8-aminooctyl)-acrylamide) (P8Am), and one anionic polymer poly (acrylic acid) (PAA) were used in the polyelectrolyte multilayer (PEM) process, based on layer-by-layer deposition [7]. PEI, one of the most potent polycationic gene delivery vectors, can efficiently complex with pDNA and facilitate endosomal release through the proton sponge effect [8]. However, PEI may cause cytotoxicity and exhibit low uptake level during its practical applications [9e11]. Conversely, P8Am, developed in our laboratory recently, could exhibit high cellular uptake efficiency with minimal toxicity [12], but could not show high transfection ability unless chloroquine [13], a well-known transfection-boosting reagent to promote endosomal escape, was incorporated in the polyplexes. Improvements in gene delivery of multilayered polyplexes may come from their mechanism of action by selecting appropriate cationic polymers and establishing the reasonable arrangement. Therefore, the purpose of this study was to develop efficient non-viral gene delivery vehicles to overcome the obstacles met in the process of transfection by sequential
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deposition of different polycations with high cellular uptake ability and endosome lytic function at different layers simultaneously. 2. Materials and methods 2.1. Preparation of binary, ternary and quaternary pDNA polyplexes The pDNA used in this study was pCMV-Luc (Promega Co., Cergy Pontoise, France) which was amplified in Escherichia coli and purified according to the supplier’s protocol (Qiagen GmbH, Hilden, Germany). PEI (branched, Mw ¼ 25,000) and PAA (Mw ¼ 15,000) are purchased from Aldrich and used without further purification. P8Am (Mw ¼ 66,700) was synthesized as described previously [12]. The pDNA vehicle constructed by PEM technique was fabricated sequentially by depositing layers of alternate polyions as schematically illustrated in Fig. 1. The binary core polyplexes of pDNA/PEI (BPI) and pDNA/P8Am (BPII) were prepared in sterile water at a weight ratio of 1/1.5 and 1/3, respectively, by adding equal volume (10 mL) of polymer solution to pDNA solution with calculated amount. Subsequently, PAA was deposited onto the positively charged core polyplexes to form ternary polyplexes at the weight ratios of 1/1.5/3 and 1/3/3 for pDNA/PEI/PAA (TPI) and pDNA/P8Am/PAA (TPII), respectively. Finally, a layer of polycation, PEI or P8Am, was deposited on the ternary polyplexes to form quaternary polyplexes TPI/P8Am, TPII/ P8Am, TPI/PEI, and TPII/PEI at outer-layer polycation/pDNA weight ratio of 1e7.
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2.2. Particle sizes and surface charges Particle sizes and surface charges of the PEM polyplexes (each containing 1 mg pDNA) were determined by a 90Plus/BI-MAS analyzer instrument (Brookhaven Instruments Corporation, Germany). The data presented reveal the average of two analyses which was performed in automatic mode using the same sample. 2.3. Agarose gel electrophoresis assay Agarose gel (1.0%, w/v) containing ethidium bromide (0.06 mg/mL, Sigma) was prepared in TAE buffer (40 mmol/L Trisacetate, 1 mmol/L EDTA). The PEM polyplexes (each containing 0.4 mg pDNA) were prepared as described above. After the polyplexes were loaded to gel electrophoresis at 100 V for 30 min and pDNA bands were visualized by using a UV transilluminator. 2.4. Cellular toxicity The cytotoxicity of the PEM polyplexes was evaluated using the MTT assay in human cervix carcinoma cell line (HeLa cell, BCRC number: 60005) and human hepatoma cell line (HepG2 cell, ATCC). In brief, cells were seeded in 96-well tissue culture plates (Costar, Cambridge, UK) at a density of 104 cells/well. Before transfection, the used medium was Dulbecco’s Modified Eagle Medium (DMEM, Gibco)
Fig. 1. (a) Schematic representation of the formation of PEM polyplexes (binary, ternary, and quaternary polyplexes). (b) Summary of symbols and sequences of PEM polyplexes.
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supplemented with high glucose and 10% fetal bovine serum (FBS, Gibco). After the cells were incubated for 24 h, the medium was replaced with 100 mL Opti-MEM medium (Gibco) containing polyplexes (each containing 0.2 mg pDNA). After incubation for another 24 h, 10 mL of (3-(4,5-dimethylthiazol-2-yl)-diphenyl) tetrazolium bromide (MTT, Sigma) in PBS (5 mg/mL) was added and incubated for 3 h at 37 C. Subsequently, the MTT solution was replaced with 50 mL of DMSO to dissolve the formazan crystal formed by live cells. The relative viability of the cells was measured by UV absorbance at 570 nm using an ELISA plate reader (SpectraMax M2e, Molecular Probe). The results were shown as a percentage of untreated cells with 100% viability. Each data was expressed as mean standard deviation of six experiments. 2.5. Cellular uptake PEM polyplexes (each containing 2 mg YOYO-1 labeled pDNA with a ratio of 1 dye molecule to 300 base pairs) were formed at desired weight ratio before transfection. Cells were plated in 6-well plates at 2.5 105 cells/well for 24 h and then the growth medium was replaced with Opti-MEM containing YOYO-1 labeled polyplexes. After incubation for 4 h, the cells were rinsed three times with PBS containing 0.5 mg/mL heparin to remove surfaceebound complexes and were collected by trypsin addition. The percentages of cells expressing YOYO-1 fluorescence were assessed by flow cytometry. The analysis was performed using a Becton Dickinson FACS flow cytometer equipped with an argon ion laser (488 nm emission wavelength) where the cell fluorescence was detected in FL1 channel. Data from 10,000 events were gated and data acquisition was performed in the linear mode, and visualized in the logarithmic mode. Dead cells were discriminated by a reduction in forward scatter and were excluded from the analysis. Each data was expressed as mean standard deviation of three experiments. 2.6. In vitro gene transfection HeLa cell and HepG2 cell were selected by using the plasmid pCMV-Luc as reporter gene for studying the gene transfection of the polyplexes. Before transfection, cells were cultured in DMEM at a density of 5 104 cells/well using 24-well tissue culture plates. After incubation for 24 h, the medium was replaced with 100 mL Opti-MEM medium containing polyplexes (each containing 1 mg pDNA). After transfection for another 24 h, cells were lysed by lysis reagent (Promega, USA) for 20 min and then centrifuged at 12,000 g at 4 C for 30 min. The relative luminescence and total protein of supernatant were determined using a luminometer (Molecular Devices/SpectraMax M5) and BCA protein assay (Pierce), respectively. Luciferase activity was indicated as relative light units per mg protein (RLU/mg protein). Each data was expressed as mean standard deviation of three experiments.
3. Results 3.1. Physicochemical properties of PEM polyplexes Fig. 2(a) and (b) shows the variation of particle sizes and surface charges of core, ternary, and quaternary pDNA polyplexes. For core polyplexes, both BPI and BPII yielded a diameter of about 135e145 nm and exhibited a positive surface charge about 45e55 mV. After the deposition of PAA onto the core polyplexes, the particle size increased slightly and the surface charge changed to negative values, which confirmed the expected charge reversal and facilitated for further opposite charge polycation incorporation. For quaternary pDNA polyplexes, the surface charge and particle size were evidently dependent on their outer-layer polycation constituents. Compared to PEI-deposited quaternary polyplexes, P8Am-deposited ones formed stable particles at higher polycation/ pDNA weight ratios, which could be attributed to the lower charge density of P8Am than PEI. It is known that addition of certain polyanions to binary polycation/DNA complexes will result in unpackaging due to competitive binding between polyanions and pDNA with polycations [14,15]. In addition, it is possible that addition of the final polycation layer in quaternary polyplexes could result in the formation of polycation/ polyanion complexes to release pDNA. To address this issue, gel electrophoresis assay was performed to check the effect of alternate polyions on polyplex integrity. As shown in Fig. 2(c), naked pDNA was detected as a band on agarose gel, but no uncomplexed pDNA was observed in any BPI, ternary TPI and quaternary TPI/P8Am polyplexes. In addition, bands of pDNA were not detected in other prepared core, ternary and quaternary polyplexes (data not shown).
Fig. 2. (a) Particle size and (b) zeta potential of PEM polyplexes. (c) Representative agarose gel electrophoresis result of BPI, TPI, and TPI/P8Am polyplexes at outer-layer polycation/pDNA weight ratio of 1e7.
3.2. Cytotoxicity of PEM polyplexes The PEM polyplexes were examined for their cytotoxicity on HeLa and HepG2 cells using MTT assay. As shown in Fig. 3(a), for HeLa cells treated with binary core polyplexes, the P8Am polyplexes showed lower cytotoxicity (91 6% cell viability) than PEI did (77 6% cell viability). Therefore, we assumed the cytotoxicity of PEI polyplexes could be improved by coating a cell-compatible polymer onto their particle surface. As expected, the viability of HeLa cells was increased when the binary PEI polyplexes were coated with PAA and further coated with P8Am. Regardless of the P8Am/pDNA ratio,
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polyplexes containing YOYO-1-labeled pDNA, followed by washing and analysis by flow cytometry. Fig. 4 shows both core polyplexes (BPI and BPII) could be taken up by HeLa and HepG2 cells, and BPII shows increased amounts of cellular uptake over BPI, consistent with our previous result that P8Am is a good candidate for the cell uptake [12]. For ternary polyplexes (TPI and TPII), as expected, no uptake of nanoparticles with negative surface charge was observed in HeLa and HepG2 cells. Interestingly, when the outer-layer polycation/pDNA weight ratio was greater than 4, coating P8Am or PEI on the ternary polyplexes could mediate higher uptake efficiency in two types of cells. Similar to cytotoxicity assay, P8Am-coated quaternary polyplexes had higher level of cell fluorescence than PEI-coated ones, regardless of their core polyplexes used.
3.4. In vitro transfection of PEM polyplexes The transfection efficiency of the core, ternary and quaternary polyplexes was analyzed by luciferase activity assay on HeLa and HepG2 cells and shown in Fig. 5. Although P8Am core polyplexes showed lower cytotoxicity and higher uptake level than PEI core polyplexes, the former did not exhibit higher luciferase activity than the later. It is known that PEI achieves enhanced transfection efficiency via the proton sponge effect [8]. Therefore, the mechanism of
Fig. 3. Cytotoxicity assay. (a) HeLa cells and (b) HepG2 cells were treated with binary, ternary, and quaternary polyplexes for 24 h. The cell viability was determined by MTT assay. The results are expressed as percentage of cell viability relative to untreated cells. Data are the mean of six experiments SD.
their viabilities were estimated as 80e95%. Similarly, other polyplexes with a P8Am surface layer such as TPII/P8Am polyplexes exhibited very low cytotoxicity. Conversely, quaternary TPI/PEI and TPII/PEI polyplexes with a PEI surface layer caused obvious cell damage and exhibited an increasing tendency of cytotoxicity with increasing the ratio of outer-layer PEI/pDNA, suggesting outer-layer PEI presented a dose-dependent effect on cytotoxicity. Therefore, compared to quaternary PEI-coated polyplexes, all of quaternary P8Am-coated polyplexes exhibited higher cell viability at all ratios, regardless of their core polyplexes used. Fig. 3(b) shows the viability of HepG2 cells treated with the prepared PEM polyplexes. Similarly, when P8Am and PEI were coated on the ternary TPI and TPII polyplexes, all of quaternary P8Am-coated polyplexes exhibited higher cell viability than PEI-coated ones at all ratios. This indicates that the P8Am layer on the PEM polyplex surface could improve the nanoparticles’ cytotoxicity. Therefore, for PEM polyplexes, the topmost-layer polycation plays an important role in governing the cytotoxic effects of polyplex properties. 3.3. Cellular uptake of PEM polyplexes To visualize the uptake of core, ternary and quaternary polyplexes, HeLa and HepG2 cells were incubated with the prepared
Fig. 4. Uptake efficiency of (a) HeLa cells and (b) HepG2 cells treated with binary, ternary, and quaternary polyplexes containing YOYO-1-labeled DNA for 24 h. Data are the mean of three experiments SD.
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4. Discussion
Fig. 5. Transfection into (a) HeLa cells and (b) HepG2 cells treated with binary, ternary, and quaternary polyplexes for 24 h. The cells were analyzed for luciferase activity. Data are the mean of three experiments SD.
action for the higher transfection efficiency of PEI core polyplexes is proposed to be due to the enhanced endosomal lysis rather than the increased cellular uptake achieved by PEI. Subsequently, HeLa and HepG2 cells were transfected with PAAcoated ternary polyplexes containing core binary polyplexes of pDNA/PEI and pDNA/P8Am to determine the polyanionic effect on transfection efficiency. Clearly, a negative surface charge caused the ternary polyplexes to have very low luciferase activity (below 103 RLU/mg protein), even PEI with the endosomal buffering entity was used to form the BPI core polyplexes. For all quaternary polyplexes, transfection efficiency gradually increased at lower outer-layer polycation/pDNA weight ratio. However, the higher ratio during transfection with PEI-coated quaternary polyplexes caused a rapid drop in luciferase activity. Conversely, the addition of more amounts of P8Am to the quaternary polyplexes maintained or even enhanced gene transfer activity. Maximum activity was observed when the quaternary polyplex contained a PEI/pDNA core and a P8Am outer layer at a P8Am/pDNA weight ratio of 5, which was 100-fold increase compared to the binary core polyplexes. The TPI/ PEI quaternary polyplexes at the outer-layer PEI/pDNA ratio of 2e5 also enhanced the transfection efficiency compared to PEI core polyplexes, but they still exhibited an approximately 10-fold drop in maximum transfection activity compared to TPI/P8Am polyplexes, regardless of HeLa or HepG2 cells.
Generally, naked pDNA is hardly transferred into cells because of enzymatic degradation and electrostatic repulsion. Thus, a variety of cationic polymers have been employed to condense and protect pDNA to efficient gene delivery [16e18]. However, the utility of cationic polymers for gene delivery application is limited by its low transfection efficiency and high cytotoxicity. Therefore, various methods have been explored to allow these cationic polymers to be conjugated with functional moieties or structurally modified to impart physicochemical properties to circumvent the obstacles met in the process of transfection, such as being able to gain entry into cell, escape the endo-lysosomal pathway, traffic through the cytoplasm, transport into nucleus and enable gene expression [19]. However, the efforts of the complicated chemical approaches are really laborious and most of the strategies designed to overcome one barrier might directly hinder the ability to overcome other barriers. Thus, rationally designing synthetic vectors with optimally balanced physicochemical properties remains extremely difficult. Provided that an admirable and convenient manner that constructs the efficient vehicle is achieved, it is a great advancement to gene therapy. The purpose of the present study is attempting to develop a new construction of gene vehicle assembled by layer-by-layer deposition, using alternate polyanions and polycations with required functionalities [7,20]. Saul and colleagues have demonstrated multilayer approaches enabled higher levels of gene expression without significant increases in cell toxicity [21]. In their studies, PEI was used to form the core polyplexes and outmost-layer constituents simultaneously. It was found that additional layer of PEI would achieve enhanced buffering capacity rather than cellular uptake, thereby enhancing transfection efficiency via the proton sponge effect. It occurred to us that using other polycations with higher uptake ability and lower toxicity as the outmost-layer constituents could serve as the basis for the rational design of a gene delivery system. Therefore, the outer layer of the vehicle was stepwise varied of weight ratios to evaluate their impact on toxicity, cellular internalization, and ultimate transfection ability. To our knowledge, this is the first report to enhance gene delivery efficiency by enhancing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability, as shown in Fig. 6. Two cationic polymers, PEI and P8Am were used in this study. The former and the later had the buffering and uptake ability, respectively, so they did not need to conjugate with various functional groups to diminish their ability to complex DNA to transfect cells. Fig. 2(a) and (b) show that all core, ternary and quaternary polyplexes through layer-by-layer opposite charge interaction were stable particles with reasonable size and surface charge. This is consistent with the previous reports that the layer thickness fabricated by PEM technique was less than 10 nm [22e24]. In addition,
Fig. 6. Schematic representation of effective PEM gene vehicle by enhancing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability.
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Fig. 2(c) shows all of the prepared PEM polyplexes retained the pDNA encapsulated in the internal of the polyplexes without disrupting pDNA interactions with the core polycations. Therefore, the successive polyelectrolyte layers are indestructible to the integrity of core polyplexes, indicating that PEM technique is a potential usage of delivery pDNA due to its simple and controllable approach. Clearly, the first point of contact for polyplex and cell membrane is the outmost polycation layer. One of the parameters reflecting the polyplex-mediated gene delivery during this contact, namely, the binding to cell surface and endocytosis, is the charge density or distribution of the outmost polycation layer, which influences the electrostatic interactions between the positively charged groups in the polycations and negatively charged proteoglycans on cell membranes, and in turn, influences whether cells can uptake without significant increase in cell toxicity. Therefore, the cellular viability and uptake expression of quaternary polyplexes were violently affected by the selection of outmost polycation layer (Figs. 3 and 4). In the previous study, we have already shown that polyacrylamide with appropriate aminooctyl side chain (P8Am) could achieve high cellular uptake efficiency and minimal cell toxicity [12]. As shown in Figs. 3 and 4, the intrinsic nature of P8Am could enhance higher cellular viability and uptake efficiency than that of PEI in binary core polyplexes. Thus, when more biocompatible P8Am was coated on the outer layer of quaternary polyplex surface, they (TPI/ P8Am and TPII/P8Am) showed higher cellular viability and uptake expression, regardless of the core polycations used. It is worth noting, compared to traditional approaches to increase cellular internalization by chemically conjugating with active moieties [25], the technique of alternating polyanion and polycation deposition to formed layered structure is a more convenient and versatile technique to construct polyplexes. For example, the ternary anionic polyplexes can be easily modified with various cationic polymers with specific characteristics to mediate internalization in a cell-specific manner. However, PEI is not recommended to coat on the outmost layer of quaternary polyplex surface (TPI/PEI and TPII/PEI), due to its obvious toxicity at higher outer-layer PEI/pDNA weight ratio. Endocytosis is known to be the major pathway through which polyplexes are taken up by cells [26]. During endocytosis, the process of endosomal escape of the gene is considered to affect the transfection efficiency strongly [27e29]. The proton sponge effect induced by polymers containing secondary and tertiary amines has been used to facilitate endosomal release [30]. It is known that PEI achieves enhanced transfection efficiency via the proton sponge effect [8]. Therefore, P8Am displayed lower luciferase activity than PEI in the binary core polyplexes, even the former showed lower cytotoxicity and higher uptake level than the later. However, it is unknown whether endosomal buffering entities should be placed in the core or on the surface layer for a multilayered polyplex. It is interesting to investigate the optimal site of PEI in the quaternary polyplexes. The mechanism of cytotoxicity of PEI is the disruption of the cell membrane by its strong positive charge density, so PEI should be placed in the core of quaternary polyplexes to prevent the direct contact between cell surface and PEI. Fig. 3 successfully proves that the cytotoxicity of binary PEI polyplexes could be improved by coating cell-compatible P8Am layer onto their particle surface. Indeed, when assayed by cellular uptake, the outer P8Am layer was required for the PEI core to ensure essentially higher cellular uptake expression with lower toxicity. Similarly, P8Am core polyplexes covered with P8Am had similar cell viability and uptake ability, but they exhibited far poorer transfection efficiencies compared to quaternary polyplexes with PEI core and outer P8Am layer (Fig. 5), indicating the importance of having PEI core for efficient transfection. Therefore, TPI/P8Am polyplexes still could facilitate endosomal release through the proposed proton sponge effect, which would not be interfered by the other polycation or polyanion layers. Consequently, the highest
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transfection efficiency achieved by TPI/P8Am polyplexes in this study can be attributed to the synergistic effect between the endosome lytic moiety (PEI) in the core and the cell-favorable polycation (P8Am) in the topmost layer to overcome the gene delivery obstacles. We optimized the amount of outer-layer P8Am and pDNA at a ratio of 5 within the quaternary polyplexes to enable the highest levels of expression with minimal toxicity. Conversely, the same composition in different sequence such as TPII/PEI polyplexes in which the cellfavorable P8Am core was externally deposited with the endosome lytic moiety (PEI), showed high toxicity and low efficiency. This indicates the reverse deposition sequence declines the transfection efficiency, thus, the PEI/pDNA/PAA/P8Am sequence for a quaternary polyplex is the same important as the polymer selection for designing safe and reliable gene delivery vehicles. 5. Conclusion In summary, this work demonstrated the advantage of preparing multilayered polyelectrolyte polyplexes for nucleic acid delivery. Especially, it is an easy technique without complicated chemical modification and without losing original functionality of polymers by layer-by-layer deposition. When the appropriate cationic polymers and the appropriate arrangement were designed to construct PEM polyplexes, they might contain cell uptake and endosome lytic functions simultaneously to express high gene transfection efficiency with reduced toxicity. References [1] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669e72. [2] Hunt KK, Vorburger SA. Gene therapy: hurdles and hopes for cancer treatment. Science 2002;297:415e6. [3] Lu B, Wang CF, Wu DQ, Li C, Zhang XZ, Zhuo RX. Chitosan based oligoamine polymers: synthesis, characterization, and gene delivery. J Control Release 2009;137:54e62. [4] Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A twostage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 2005;11:990e5. [5] Kurosaki T, Kitahara T, Fumoto S, Nishida K, Nakamura J, Niidome T, et al. Ternary complexes of pDNA, polyethylenimine, and g-polyglutamic acid for gene delivery systems. Biomaterials 2009;30:2846e53. [6] Chung YC, Hsieh WY, Young TH. Polycation/DNA complexes coated with oligonucleotides for gene delivery. Biomaterials 2010;31:4194e203. [7] Decher G. Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science 1997;277:1232e7. [8] Behr JP. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 1997;51:34e6. [9] Merdan T, Callahan J, Petersen H, Kunath K, Bakowsky U, Kopeckova P, et al. Pegylated polyethylenimine-Fab’ antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells. Bioconjug Chem 2003;14:989e96. [10] Gabrielson NP, Pack DW. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules 2006;7:2427e35. [11] Yang Y, Zhang Z, Chen L, Gu W, Li Y. Galactosylated poly(2-(2-aminoethyoxy) ethoxy)phosphazene/DNA complex nanoparticles: in vitro and in vivo evaluation for gene delivery. Biomacromolecules 2010;11:927e33. [12] Ke JH, Wei MF, Shieh MJ, Young TH. Design, synthesis and evaluation of cationic poly (N-substituent acrylamide)s for in vitro gene delivery. J Biomater Sci Polym Ed. doi:10.1163/092050610X502767, in press. [13] Eldred SE, Pancost MR, Otte KM, Rozema D, Stahl SS, Gellman SH. Effects of side chain configuration and backbone spacing on the gene delivery properties of lysine-derived cationic polymers. Bioconjug Chem 2005;16:694e9. [14] Xu Y, Szoka FC. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 1996;35:5616e23. [15] Zelikin AN, Trukhanova ES, Putnam D, Izumrudov VA, Litmanovich AA. Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes. J Am Chem Soc 2003;125:13693e9. [16] Nimesh S, Goyal A, Pawar V, Jayaraman S, Kumar P, Chandra R, et al. Polyethylenimine nanoparticles as efficient transfecting agents for mammalian cells. J Control Release 2006;110:457e68. [17] Kwoh DY, Coffin CC, Lollo CP, Jovenal J, Banaszczyk MG, Mullen P, et al. Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver. Biochim Biophys Acta 1999;1444:171e90. Gene Struct Expression.
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Multilayered polyplexes with the endosomal buffering polycation in the core and the cell uptake-favorable polycation in the outer layer for enhanced gene delivery Jin-He Ke a, Tai-Horng Young a, b, * a b
Institute of Polymer Science and Engineering, National Taiwan University, Taipei, 106, Taiwan, ROC Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2010 Accepted 18 August 2010 Available online 22 September 2010
In the present study, quaternary polyplexes were prepared by sequential addition of polycations (polyethylenimine (PEI) or poly (N-(8-aminooctyl)-acrylamide) (P8Am)) for loading pDNA into the core polyplexes and poly (acrylic acid) (PAA) for reversing charges to deposit additional polycation (PEI or P8Am) layer. It was found the cytotoxicity and cellular uptake expression of PEI core polyplexes could be improved by coating a cell uptake-favorable P8Am layer. Conversely, P8Am could not facilitate endosomal release through the proposed proton sponge effect so the PEI core was required for the P8Amcoated quaternary polyplexes to ensure efficient transfection. Consequently, an efficient and safe non-viral gene vehicle was constructed by layer-by-layer deposition, using alternate polyanion and polycation with required functionalities to overcome the obstacles met in the process of transfection. Maximum transfection activity with minimal toxicity was observed when the quaternary polyplex of pDNA/PEI/PAA/P8Am was prepared at a weight ratio of 1/1.5/3/5. Conversely, the same composition in different position such as the cell-favorable P8Am core was externally deposited with the endosome lytic moiety, PEI showed high toxicity and low efficiency. This indicates the pDNA/PEI/PAA/P8Am sequence for a quaternary polyplex is as important as the functional polymer selection for designing safe and reliable gene delivery vehicles. We demonstrate here that gene delivery efficiency may be improved by increasing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Gene therapy Gene vehicle Polyelectrolyte multilayer Polycation
1. Introduction Gene therapy offers a potential method to treat disease ranging from inherited disorders to acquired conditions and cancer by transferring exogenous nucleic acids into cells to alter protein expression profiles [1,2]. Generally, cationic nanoparticles would be appropriately applied to cellular uptake because of the electrostatic interaction between nanoparticles and cellular membrane, but it is limited by its low transfection efficiency and high cytotoxicity [3,4]. Except decreasing positive charge by chemical modification, “recharging” of polyplexes using polyanions to reverse the surface charge of polyplexes has been demonstrated to decrease the cytotoxicity of polycation/DNA complexes [5]. We have previously described the formation of ternary polyplexes containing pDNA, synthetic polycations and polyanions [6]. In this study, we extended
* Corresponding author. Institute of Biomedical Engineering, College of Medicine, National Taiwan University, Taipei 100, Taiwan. Tel.: þ886 2 23123456x81455; fax: þ886 2 23940049. E-mail address: [email protected] (T.-H. Young). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.08.066
these studies and found that the further addition of a polycationic layer on the ternary polyplexes could increase the levels of gene expression with reduced toxicity. Two cationic polymers, polyethylenimine (PEI) and poly (N-(8-aminooctyl)-acrylamide) (P8Am), and one anionic polymer poly (acrylic acid) (PAA) were used in the polyelectrolyte multilayer (PEM) process, based on layer-by-layer deposition [7]. PEI, one of the most potent polycationic gene delivery vectors, can efficiently complex with pDNA and facilitate endosomal release through the proton sponge effect [8]. However, PEI may cause cytotoxicity and exhibit low uptake level during its practical applications [9e11]. Conversely, P8Am, developed in our laboratory recently, could exhibit high cellular uptake efficiency with minimal toxicity [12], but could not show high transfection ability unless chloroquine [13], a well-known transfection-boosting reagent to promote endosomal escape, was incorporated in the polyplexes. Improvements in gene delivery of multilayered polyplexes may come from their mechanism of action by selecting appropriate cationic polymers and establishing the reasonable arrangement. Therefore, the purpose of this study was to develop efficient non-viral gene delivery vehicles to overcome the obstacles met in the process of transfection by sequential
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deposition of different polycations with high cellular uptake ability and endosome lytic function at different layers simultaneously. 2. Materials and methods 2.1. Preparation of binary, ternary and quaternary pDNA polyplexes The pDNA used in this study was pCMV-Luc (Promega Co., Cergy Pontoise, France) which was amplified in Escherichia coli and purified according to the supplier’s protocol (Qiagen GmbH, Hilden, Germany). PEI (branched, Mw ¼ 25,000) and PAA (Mw ¼ 15,000) are purchased from Aldrich and used without further purification. P8Am (Mw ¼ 66,700) was synthesized as described previously [12]. The pDNA vehicle constructed by PEM technique was fabricated sequentially by depositing layers of alternate polyions as schematically illustrated in Fig. 1. The binary core polyplexes of pDNA/PEI (BPI) and pDNA/P8Am (BPII) were prepared in sterile water at a weight ratio of 1/1.5 and 1/3, respectively, by adding equal volume (10 mL) of polymer solution to pDNA solution with calculated amount. Subsequently, PAA was deposited onto the positively charged core polyplexes to form ternary polyplexes at the weight ratios of 1/1.5/3 and 1/3/3 for pDNA/PEI/PAA (TPI) and pDNA/P8Am/PAA (TPII), respectively. Finally, a layer of polycation, PEI or P8Am, was deposited on the ternary polyplexes to form quaternary polyplexes TPI/P8Am, TPII/ P8Am, TPI/PEI, and TPII/PEI at outer-layer polycation/pDNA weight ratio of 1e7.
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2.2. Particle sizes and surface charges Particle sizes and surface charges of the PEM polyplexes (each containing 1 mg pDNA) were determined by a 90Plus/BI-MAS analyzer instrument (Brookhaven Instruments Corporation, Germany). The data presented reveal the average of two analyses which was performed in automatic mode using the same sample. 2.3. Agarose gel electrophoresis assay Agarose gel (1.0%, w/v) containing ethidium bromide (0.06 mg/mL, Sigma) was prepared in TAE buffer (40 mmol/L Trisacetate, 1 mmol/L EDTA). The PEM polyplexes (each containing 0.4 mg pDNA) were prepared as described above. After the polyplexes were loaded to gel electrophoresis at 100 V for 30 min and pDNA bands were visualized by using a UV transilluminator. 2.4. Cellular toxicity The cytotoxicity of the PEM polyplexes was evaluated using the MTT assay in human cervix carcinoma cell line (HeLa cell, BCRC number: 60005) and human hepatoma cell line (HepG2 cell, ATCC). In brief, cells were seeded in 96-well tissue culture plates (Costar, Cambridge, UK) at a density of 104 cells/well. Before transfection, the used medium was Dulbecco’s Modified Eagle Medium (DMEM, Gibco)
Fig. 1. (a) Schematic representation of the formation of PEM polyplexes (binary, ternary, and quaternary polyplexes). (b) Summary of symbols and sequences of PEM polyplexes.
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supplemented with high glucose and 10% fetal bovine serum (FBS, Gibco). After the cells were incubated for 24 h, the medium was replaced with 100 mL Opti-MEM medium (Gibco) containing polyplexes (each containing 0.2 mg pDNA). After incubation for another 24 h, 10 mL of (3-(4,5-dimethylthiazol-2-yl)-diphenyl) tetrazolium bromide (MTT, Sigma) in PBS (5 mg/mL) was added and incubated for 3 h at 37 C. Subsequently, the MTT solution was replaced with 50 mL of DMSO to dissolve the formazan crystal formed by live cells. The relative viability of the cells was measured by UV absorbance at 570 nm using an ELISA plate reader (SpectraMax M2e, Molecular Probe). The results were shown as a percentage of untreated cells with 100% viability. Each data was expressed as mean standard deviation of six experiments. 2.5. Cellular uptake PEM polyplexes (each containing 2 mg YOYO-1 labeled pDNA with a ratio of 1 dye molecule to 300 base pairs) were formed at desired weight ratio before transfection. Cells were plated in 6-well plates at 2.5 105 cells/well for 24 h and then the growth medium was replaced with Opti-MEM containing YOYO-1 labeled polyplexes. After incubation for 4 h, the cells were rinsed three times with PBS containing 0.5 mg/mL heparin to remove surfaceebound complexes and were collected by trypsin addition. The percentages of cells expressing YOYO-1 fluorescence were assessed by flow cytometry. The analysis was performed using a Becton Dickinson FACS flow cytometer equipped with an argon ion laser (488 nm emission wavelength) where the cell fluorescence was detected in FL1 channel. Data from 10,000 events were gated and data acquisition was performed in the linear mode, and visualized in the logarithmic mode. Dead cells were discriminated by a reduction in forward scatter and were excluded from the analysis. Each data was expressed as mean standard deviation of three experiments. 2.6. In vitro gene transfection HeLa cell and HepG2 cell were selected by using the plasmid pCMV-Luc as reporter gene for studying the gene transfection of the polyplexes. Before transfection, cells were cultured in DMEM at a density of 5 104 cells/well using 24-well tissue culture plates. After incubation for 24 h, the medium was replaced with 100 mL Opti-MEM medium containing polyplexes (each containing 1 mg pDNA). After transfection for another 24 h, cells were lysed by lysis reagent (Promega, USA) for 20 min and then centrifuged at 12,000 g at 4 C for 30 min. The relative luminescence and total protein of supernatant were determined using a luminometer (Molecular Devices/SpectraMax M5) and BCA protein assay (Pierce), respectively. Luciferase activity was indicated as relative light units per mg protein (RLU/mg protein). Each data was expressed as mean standard deviation of three experiments.
3. Results 3.1. Physicochemical properties of PEM polyplexes Fig. 2(a) and (b) shows the variation of particle sizes and surface charges of core, ternary, and quaternary pDNA polyplexes. For core polyplexes, both BPI and BPII yielded a diameter of about 135e145 nm and exhibited a positive surface charge about 45e55 mV. After the deposition of PAA onto the core polyplexes, the particle size increased slightly and the surface charge changed to negative values, which confirmed the expected charge reversal and facilitated for further opposite charge polycation incorporation. For quaternary pDNA polyplexes, the surface charge and particle size were evidently dependent on their outer-layer polycation constituents. Compared to PEI-deposited quaternary polyplexes, P8Am-deposited ones formed stable particles at higher polycation/ pDNA weight ratios, which could be attributed to the lower charge density of P8Am than PEI. It is known that addition of certain polyanions to binary polycation/DNA complexes will result in unpackaging due to competitive binding between polyanions and pDNA with polycations [14,15]. In addition, it is possible that addition of the final polycation layer in quaternary polyplexes could result in the formation of polycation/ polyanion complexes to release pDNA. To address this issue, gel electrophoresis assay was performed to check the effect of alternate polyions on polyplex integrity. As shown in Fig. 2(c), naked pDNA was detected as a band on agarose gel, but no uncomplexed pDNA was observed in any BPI, ternary TPI and quaternary TPI/P8Am polyplexes. In addition, bands of pDNA were not detected in other prepared core, ternary and quaternary polyplexes (data not shown).
Fig. 2. (a) Particle size and (b) zeta potential of PEM polyplexes. (c) Representative agarose gel electrophoresis result of BPI, TPI, and TPI/P8Am polyplexes at outer-layer polycation/pDNA weight ratio of 1e7.
3.2. Cytotoxicity of PEM polyplexes The PEM polyplexes were examined for their cytotoxicity on HeLa and HepG2 cells using MTT assay. As shown in Fig. 3(a), for HeLa cells treated with binary core polyplexes, the P8Am polyplexes showed lower cytotoxicity (91 6% cell viability) than PEI did (77 6% cell viability). Therefore, we assumed the cytotoxicity of PEI polyplexes could be improved by coating a cell-compatible polymer onto their particle surface. As expected, the viability of HeLa cells was increased when the binary PEI polyplexes were coated with PAA and further coated with P8Am. Regardless of the P8Am/pDNA ratio,
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polyplexes containing YOYO-1-labeled pDNA, followed by washing and analysis by flow cytometry. Fig. 4 shows both core polyplexes (BPI and BPII) could be taken up by HeLa and HepG2 cells, and BPII shows increased amounts of cellular uptake over BPI, consistent with our previous result that P8Am is a good candidate for the cell uptake [12]. For ternary polyplexes (TPI and TPII), as expected, no uptake of nanoparticles with negative surface charge was observed in HeLa and HepG2 cells. Interestingly, when the outer-layer polycation/pDNA weight ratio was greater than 4, coating P8Am or PEI on the ternary polyplexes could mediate higher uptake efficiency in two types of cells. Similar to cytotoxicity assay, P8Am-coated quaternary polyplexes had higher level of cell fluorescence than PEI-coated ones, regardless of their core polyplexes used.
3.4. In vitro transfection of PEM polyplexes The transfection efficiency of the core, ternary and quaternary polyplexes was analyzed by luciferase activity assay on HeLa and HepG2 cells and shown in Fig. 5. Although P8Am core polyplexes showed lower cytotoxicity and higher uptake level than PEI core polyplexes, the former did not exhibit higher luciferase activity than the later. It is known that PEI achieves enhanced transfection efficiency via the proton sponge effect [8]. Therefore, the mechanism of
Fig. 3. Cytotoxicity assay. (a) HeLa cells and (b) HepG2 cells were treated with binary, ternary, and quaternary polyplexes for 24 h. The cell viability was determined by MTT assay. The results are expressed as percentage of cell viability relative to untreated cells. Data are the mean of six experiments SD.
their viabilities were estimated as 80e95%. Similarly, other polyplexes with a P8Am surface layer such as TPII/P8Am polyplexes exhibited very low cytotoxicity. Conversely, quaternary TPI/PEI and TPII/PEI polyplexes with a PEI surface layer caused obvious cell damage and exhibited an increasing tendency of cytotoxicity with increasing the ratio of outer-layer PEI/pDNA, suggesting outer-layer PEI presented a dose-dependent effect on cytotoxicity. Therefore, compared to quaternary PEI-coated polyplexes, all of quaternary P8Am-coated polyplexes exhibited higher cell viability at all ratios, regardless of their core polyplexes used. Fig. 3(b) shows the viability of HepG2 cells treated with the prepared PEM polyplexes. Similarly, when P8Am and PEI were coated on the ternary TPI and TPII polyplexes, all of quaternary P8Am-coated polyplexes exhibited higher cell viability than PEI-coated ones at all ratios. This indicates that the P8Am layer on the PEM polyplex surface could improve the nanoparticles’ cytotoxicity. Therefore, for PEM polyplexes, the topmost-layer polycation plays an important role in governing the cytotoxic effects of polyplex properties. 3.3. Cellular uptake of PEM polyplexes To visualize the uptake of core, ternary and quaternary polyplexes, HeLa and HepG2 cells were incubated with the prepared
Fig. 4. Uptake efficiency of (a) HeLa cells and (b) HepG2 cells treated with binary, ternary, and quaternary polyplexes containing YOYO-1-labeled DNA for 24 h. Data are the mean of three experiments SD.
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4. Discussion
Fig. 5. Transfection into (a) HeLa cells and (b) HepG2 cells treated with binary, ternary, and quaternary polyplexes for 24 h. The cells were analyzed for luciferase activity. Data are the mean of three experiments SD.
action for the higher transfection efficiency of PEI core polyplexes is proposed to be due to the enhanced endosomal lysis rather than the increased cellular uptake achieved by PEI. Subsequently, HeLa and HepG2 cells were transfected with PAAcoated ternary polyplexes containing core binary polyplexes of pDNA/PEI and pDNA/P8Am to determine the polyanionic effect on transfection efficiency. Clearly, a negative surface charge caused the ternary polyplexes to have very low luciferase activity (below 103 RLU/mg protein), even PEI with the endosomal buffering entity was used to form the BPI core polyplexes. For all quaternary polyplexes, transfection efficiency gradually increased at lower outer-layer polycation/pDNA weight ratio. However, the higher ratio during transfection with PEI-coated quaternary polyplexes caused a rapid drop in luciferase activity. Conversely, the addition of more amounts of P8Am to the quaternary polyplexes maintained or even enhanced gene transfer activity. Maximum activity was observed when the quaternary polyplex contained a PEI/pDNA core and a P8Am outer layer at a P8Am/pDNA weight ratio of 5, which was 100-fold increase compared to the binary core polyplexes. The TPI/ PEI quaternary polyplexes at the outer-layer PEI/pDNA ratio of 2e5 also enhanced the transfection efficiency compared to PEI core polyplexes, but they still exhibited an approximately 10-fold drop in maximum transfection activity compared to TPI/P8Am polyplexes, regardless of HeLa or HepG2 cells.
Generally, naked pDNA is hardly transferred into cells because of enzymatic degradation and electrostatic repulsion. Thus, a variety of cationic polymers have been employed to condense and protect pDNA to efficient gene delivery [16e18]. However, the utility of cationic polymers for gene delivery application is limited by its low transfection efficiency and high cytotoxicity. Therefore, various methods have been explored to allow these cationic polymers to be conjugated with functional moieties or structurally modified to impart physicochemical properties to circumvent the obstacles met in the process of transfection, such as being able to gain entry into cell, escape the endo-lysosomal pathway, traffic through the cytoplasm, transport into nucleus and enable gene expression [19]. However, the efforts of the complicated chemical approaches are really laborious and most of the strategies designed to overcome one barrier might directly hinder the ability to overcome other barriers. Thus, rationally designing synthetic vectors with optimally balanced physicochemical properties remains extremely difficult. Provided that an admirable and convenient manner that constructs the efficient vehicle is achieved, it is a great advancement to gene therapy. The purpose of the present study is attempting to develop a new construction of gene vehicle assembled by layer-by-layer deposition, using alternate polyanions and polycations with required functionalities [7,20]. Saul and colleagues have demonstrated multilayer approaches enabled higher levels of gene expression without significant increases in cell toxicity [21]. In their studies, PEI was used to form the core polyplexes and outmost-layer constituents simultaneously. It was found that additional layer of PEI would achieve enhanced buffering capacity rather than cellular uptake, thereby enhancing transfection efficiency via the proton sponge effect. It occurred to us that using other polycations with higher uptake ability and lower toxicity as the outmost-layer constituents could serve as the basis for the rational design of a gene delivery system. Therefore, the outer layer of the vehicle was stepwise varied of weight ratios to evaluate their impact on toxicity, cellular internalization, and ultimate transfection ability. To our knowledge, this is the first report to enhance gene delivery efficiency by enhancing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability, as shown in Fig. 6. Two cationic polymers, PEI and P8Am were used in this study. The former and the later had the buffering and uptake ability, respectively, so they did not need to conjugate with various functional groups to diminish their ability to complex DNA to transfect cells. Fig. 2(a) and (b) show that all core, ternary and quaternary polyplexes through layer-by-layer opposite charge interaction were stable particles with reasonable size and surface charge. This is consistent with the previous reports that the layer thickness fabricated by PEM technique was less than 10 nm [22e24]. In addition,
Fig. 6. Schematic representation of effective PEM gene vehicle by enhancing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability.
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Fig. 2(c) shows all of the prepared PEM polyplexes retained the pDNA encapsulated in the internal of the polyplexes without disrupting pDNA interactions with the core polycations. Therefore, the successive polyelectrolyte layers are indestructible to the integrity of core polyplexes, indicating that PEM technique is a potential usage of delivery pDNA due to its simple and controllable approach. Clearly, the first point of contact for polyplex and cell membrane is the outmost polycation layer. One of the parameters reflecting the polyplex-mediated gene delivery during this contact, namely, the binding to cell surface and endocytosis, is the charge density or distribution of the outmost polycation layer, which influences the electrostatic interactions between the positively charged groups in the polycations and negatively charged proteoglycans on cell membranes, and in turn, influences whether cells can uptake without significant increase in cell toxicity. Therefore, the cellular viability and uptake expression of quaternary polyplexes were violently affected by the selection of outmost polycation layer (Figs. 3 and 4). In the previous study, we have already shown that polyacrylamide with appropriate aminooctyl side chain (P8Am) could achieve high cellular uptake efficiency and minimal cell toxicity [12]. As shown in Figs. 3 and 4, the intrinsic nature of P8Am could enhance higher cellular viability and uptake efficiency than that of PEI in binary core polyplexes. Thus, when more biocompatible P8Am was coated on the outer layer of quaternary polyplex surface, they (TPI/ P8Am and TPII/P8Am) showed higher cellular viability and uptake expression, regardless of the core polycations used. It is worth noting, compared to traditional approaches to increase cellular internalization by chemically conjugating with active moieties [25], the technique of alternating polyanion and polycation deposition to formed layered structure is a more convenient and versatile technique to construct polyplexes. For example, the ternary anionic polyplexes can be easily modified with various cationic polymers with specific characteristics to mediate internalization in a cell-specific manner. However, PEI is not recommended to coat on the outmost layer of quaternary polyplex surface (TPI/PEI and TPII/PEI), due to its obvious toxicity at higher outer-layer PEI/pDNA weight ratio. Endocytosis is known to be the major pathway through which polyplexes are taken up by cells [26]. During endocytosis, the process of endosomal escape of the gene is considered to affect the transfection efficiency strongly [27e29]. The proton sponge effect induced by polymers containing secondary and tertiary amines has been used to facilitate endosomal release [30]. It is known that PEI achieves enhanced transfection efficiency via the proton sponge effect [8]. Therefore, P8Am displayed lower luciferase activity than PEI in the binary core polyplexes, even the former showed lower cytotoxicity and higher uptake level than the later. However, it is unknown whether endosomal buffering entities should be placed in the core or on the surface layer for a multilayered polyplex. It is interesting to investigate the optimal site of PEI in the quaternary polyplexes. The mechanism of cytotoxicity of PEI is the disruption of the cell membrane by its strong positive charge density, so PEI should be placed in the core of quaternary polyplexes to prevent the direct contact between cell surface and PEI. Fig. 3 successfully proves that the cytotoxicity of binary PEI polyplexes could be improved by coating cell-compatible P8Am layer onto their particle surface. Indeed, when assayed by cellular uptake, the outer P8Am layer was required for the PEI core to ensure essentially higher cellular uptake expression with lower toxicity. Similarly, P8Am core polyplexes covered with P8Am had similar cell viability and uptake ability, but they exhibited far poorer transfection efficiencies compared to quaternary polyplexes with PEI core and outer P8Am layer (Fig. 5), indicating the importance of having PEI core for efficient transfection. Therefore, TPI/P8Am polyplexes still could facilitate endosomal release through the proposed proton sponge effect, which would not be interfered by the other polycation or polyanion layers. Consequently, the highest
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transfection efficiency achieved by TPI/P8Am polyplexes in this study can be attributed to the synergistic effect between the endosome lytic moiety (PEI) in the core and the cell-favorable polycation (P8Am) in the topmost layer to overcome the gene delivery obstacles. We optimized the amount of outer-layer P8Am and pDNA at a ratio of 5 within the quaternary polyplexes to enable the highest levels of expression with minimal toxicity. Conversely, the same composition in different sequence such as TPII/PEI polyplexes in which the cellfavorable P8Am core was externally deposited with the endosome lytic moiety (PEI), showed high toxicity and low efficiency. This indicates the reverse deposition sequence declines the transfection efficiency, thus, the PEI/pDNA/PAA/P8Am sequence for a quaternary polyplex is the same important as the polymer selection for designing safe and reliable gene delivery vehicles. 5. Conclusion In summary, this work demonstrated the advantage of preparing multilayered polyelectrolyte polyplexes for nucleic acid delivery. Especially, it is an easy technique without complicated chemical modification and without losing original functionality of polymers by layer-by-layer deposition. When the appropriate cationic polymers and the appropriate arrangement were designed to construct PEM polyplexes, they might contain cell uptake and endosome lytic functions simultaneously to express high gene transfection efficiency with reduced toxicity. References [1] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000;288:669e72. [2] Hunt KK, Vorburger SA. Gene therapy: hurdles and hopes for cancer treatment. Science 2002;297:415e6. [3] Lu B, Wang CF, Wu DQ, Li C, Zhang XZ, Zhuo RX. Chitosan based oligoamine polymers: synthesis, characterization, and gene delivery. J Control Release 2009;137:54e62. [4] Moghimi SM, Symonds P, Murray JC, Hunter AC, Debska G, Szewczyk A. A twostage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol Ther 2005;11:990e5. [5] Kurosaki T, Kitahara T, Fumoto S, Nishida K, Nakamura J, Niidome T, et al. Ternary complexes of pDNA, polyethylenimine, and g-polyglutamic acid for gene delivery systems. Biomaterials 2009;30:2846e53. [6] Chung YC, Hsieh WY, Young TH. Polycation/DNA complexes coated with oligonucleotides for gene delivery. Biomaterials 2010;31:4194e203. [7] Decher G. Fuzzy nanoassemblies: toward layered polymeric multicomposites. Science 1997;277:1232e7. [8] Behr JP. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia 1997;51:34e6. [9] Merdan T, Callahan J, Petersen H, Kunath K, Bakowsky U, Kopeckova P, et al. Pegylated polyethylenimine-Fab’ antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells. Bioconjug Chem 2003;14:989e96. [10] Gabrielson NP, Pack DW. Acetylation of polyethylenimine enhances gene delivery via weakened polymer/DNA interactions. Biomacromolecules 2006;7:2427e35. [11] Yang Y, Zhang Z, Chen L, Gu W, Li Y. Galactosylated poly(2-(2-aminoethyoxy) ethoxy)phosphazene/DNA complex nanoparticles: in vitro and in vivo evaluation for gene delivery. Biomacromolecules 2010;11:927e33. [12] Ke JH, Wei MF, Shieh MJ, Young TH. Design, synthesis and evaluation of cationic poly (N-substituent acrylamide)s for in vitro gene delivery. J Biomater Sci Polym Ed. doi:10.1163/092050610X502767, in press. [13] Eldred SE, Pancost MR, Otte KM, Rozema D, Stahl SS, Gellman SH. Effects of side chain configuration and backbone spacing on the gene delivery properties of lysine-derived cationic polymers. Bioconjug Chem 2005;16:694e9. [14] Xu Y, Szoka FC. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry 1996;35:5616e23. [15] Zelikin AN, Trukhanova ES, Putnam D, Izumrudov VA, Litmanovich AA. Competitive reactions in solutions of poly-L-histidine, calf thymus DNA, and synthetic polyanions: determining the binding constants of polyelectrolytes. J Am Chem Soc 2003;125:13693e9. [16] Nimesh S, Goyal A, Pawar V, Jayaraman S, Kumar P, Chandra R, et al. Polyethylenimine nanoparticles as efficient transfecting agents for mammalian cells. J Control Release 2006;110:457e68. [17] Kwoh DY, Coffin CC, Lollo CP, Jovenal J, Banaszczyk MG, Mullen P, et al. Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver. Biochim Biophys Acta 1999;1444:171e90. Gene Struct Expression.
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