CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201402293
Bioreducible Guanidinylated Polyethylenimine for Efficient Gene Delivery Duhwan Lee,[a] Yeong Mi Lee,[a] Cherlhyun Jeong,[b] Jun Lee,[c] and Won Jong Kim*[a] Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought-after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI-SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI-SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI-SS demonstrate that it has the appropriate size (~ 200 nm) and surface potential (~ 30 mV) at
a nitrogen-to-phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI-SS, GBPEI-SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.
Introduction Nonviral gene delivery systems can overcome the demerits of viral gene delivery systems, such as the host immune response, low capacity for genes, risk of replication, and random DNA insertion.[1] Several nonviral gene delivery systems use cationic polymers, liposomes, self-assembled nanoparticles or inorganic nanoparticles.[2] These nonviral vectors have advantages of facile synthesis and modification, non-immunogenicity, large capacity for genes, and low cytotoxicity.[3] Polymeric gene delivery systems are suitable candidates for efficient gene delivery due to the spontaneous formation of a nano-sized complex (polyplex) with negatively charged DNA by electrostatic interaction, the efficient internalization into the cell, and the easy endosomal escape.[4] Branched and linear polyethylenimine (PEI), chitosan, poly-llysine, and many cationic polymers have been developed to deliver DNA to cells. Branched PEI (BPEI) is one of the most effective cationic polymers and is regarded as the standard to [a] D. Lee,+ Dr. Y. M. Lee,+ Prof. Dr. W. J. Kim Center for Self-assembly and Complexity, Institute for Basic Science and Department of Chemistry Pohang University of Science and Technology (POSTECH) San 31, Hyoja-dong, Pohang 790-784 (Republic of Korea) E-mail: [email protected] [b] Dr. C. Jeong Center for Theragnosis, Biomedical Research Institute Korea Institute of Science and Technology (KIST) Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 136-791 (Republic of Korea) [c] Dr. J. Lee Department of Oral & Maxillofacial Surgery, School of dentistry Wonkwang Bone Regeneration Research Institute Wonkwang University, Iksan 570-749 (Republic of Korea) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402293.
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which polymeric gene delivery systems should be compared. However, the transfection efficiency of BPEI depends on its molecular weight (MW) along with its nonspecific cytotoxicity. BPEI with high MW (> 25 kDa) shows high transfection efficiency with significant cytotoxicity, whereas BPEI with low MW (< 1.8 kDa) shows low transfection efficiency with low cytotoxicity.[5] To obtain high transfection efficiency and low cytotoxicity, researchers have introduced the strategy of using bioreducible disulfide linkage to crosslink low MW BPEI.[6] In the extracellular compartments, this bioreducible polymer forms stable polyplexes due to low concentrations of reducing agents such as glutathione. However, in intracellular compartments, disulfide bonds can be reduced and cleaved to thiol groups due to the reducing environment of cytosolic compartments. As a result, bioreducible crosslinked BPEI can be degraded to low MW BPEI, which releases its DNA contents efficiently.[6d, 7] However, to increase gene transfection efficiency, the cellular uptake of crosslinked BPEI into cells remains to be optimized. To achieve this goal, we were inspired by the functionality of cell-penetrating peptides (CPPs). CPPs, short peptides of 30 or fewer amino acids, facilitate uptake of various molecular cargos into cells. Therefore, conjugation of the carriers with CPPs such as HIV-1 Tat peptide and Drosophila antennapedia homoprotein can enhance cellular uptake, and thus increase the delivery efficiency. CPPs have cationic or amphiphilic amino acid moieties, such as arginine and lysine residues, in their sequence.[8] Arginine residues of CPPs contribute strongly to their high internalization into cells.[9] The guanidine functional group of the arginine residue can interact with phosphate, sulfate or carboxylate of the proteoglycans on the cell surface by bidentate hydrogen bonding.[10] Inspired by this strategy, researchers have reported guanidinylated polymeric gene delivery systems that use poly(allylChemMedChem 2014, 9, 2718 – 2724
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CHEMMEDCHEM FULL PAPERS amine), polyethylenimine–polyoxypropylene–polyoxyethylene, cationic polymethacrylate, or poly(cystaminebisacrylamide-diaminohexane). These guanidinylated polymers show high gene transfection efficiency and improved intracellular uptake over nonguanidinylated analogues.[11] Therefore, we expect that conjugation of a guanidine group to bioreducible polymers will increase their gene delivery efficiency by increasing cellular uptake and the efficiency of gene release. Moreover, conjugation of the guanidine group to a BPEI can be expected to decrease its cytotoxicity by converting the primary amine to a guanidine group (Figure 1). In this study, guanidinylated disulfide crosslinked BPEI (GBPEI-SS) was synthesized, and the physicochemical properties of the polyplexes were investigated. Transfection efficiency, cytotoxicity and cellular uptake efficiency were also evaluated.
Figure 1. Schematic diagram of gene delivery based on the bioreducible guanidinylated branched polyethylenimine (GBPEI-SS) polyplex.
Results and Discussion
www.chemmedchem.org Table 1. Characterization of nonguanidinylated (BPEI-SS) and guanidinylated (GBPEI-SS) bioreducible branched polyethylenimine. Denotation
BPEI-SS GBPEI-SS4x GBPEI-SS8x
Thiol group Feed ratio[a] Degree of thiolation[b] 7 7 7
6.55 5.42 4.58
Guanidine group Feed ratio[c] Degree of guanidinylation[d] – 4 8
– 2.4 (34.4) 4.3 (61.6)
[a] Propylene sulfide/BPEI1.2K molar ratio. [b] Total quantities of thiol in one chain of BPEI1.2K, measured by 1H NMR. [c] 2-Ethyl-2-thiopsuedourea hydrobromide/BPEI1.2K molar ratio. [d] Total quantity of guanidine in one chain of BPEI1.2K, measured by elemental analysis. Values in parentheses are the percentage of primary amines converted into guanidine group.
termined by comparing the C/N ratio between BPEI1.2K and GBPEI. Using four and eight equivalents of 2-ethyl-2-thiopseudourea gave a conjugation ratio of 2.4 (GBPEI4x) and 4.3 (GBPEI8x), respectively (Table 1). GBPEI was endowed with bioreducibility by using a disulfide bond to crosslink it. Using propylene sulfide as a thiolation reagent (7 equiv to BPEI1.2K), thiol groups were conjugated to primary and secondary amine groups of GBPEI. During the reaction, the buffering capacity of GBPEI did not change significantly because primary amine and secondary amine were transformed to higher order amine groups.[6d] The degree of thiolation was determined using 1H NMR. The calculated conjugation ratios were 5.42 and 4.58 for GBPEI-SS4x and GBPEI-SS8x, respectively. As a control, bioreducible BPEI with no guanidine group (BPEI-SS) was also synthesized. The degree of thiolation of BPEI-SS was 6.55. The successful synthesis of GBPEI-SS was also confirmed using 1H NMR. In the 1H NMR spectra, a methyl proton adjacent to thiol (d = 1.2–1.5 ppm), methylene proton of BPEI (d = 2.5– 3.2 ppm) and methylene proton adjacent to guanidine group (d = 3.2–3.8 ppm) appeared.
Synthesis and characterization of GBPEI-SS For the synthesis of GBPEI-SS (Scheme 1), a guanidine group was first introduced to low MW BPEI1.2K. To achieve this, 2ethyl-2-thiopseudourea hydrobromide was used as a guanidinylation agent at different molar ratios (4 and 8 equiv). During the reaction, ethanethiol was generated as a byproduct through attack of the thiopseudourea carbon by the primary amine group of BPEI. After purification by dialysis, elemental analysis was used to confirm that a guanidine group had been introduced to BPEI. The conjugation ratio of guanidine was de-
Scheme 1. Preparation of bioreducible guanidinylated branched polyethylenimine (GBPEI-SS). Reagents and conditions: a) MeOH, RT, 24 h; b) 1. propylene sulfide, MeOH, 60 8C, 24 h; 2. DMSO, RT, 48 h.
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Physicochemical property of polyplexes For efficient gene delivery, the cationic polymer that forms the polyplex with plasmid DNA (pDNA) should condense pDNA effectively. An agarose gel retardation assay was conducted to investigate the formation of the GBPEI-SS/pDNA complex at different N/P ratios (where N is the molar amount of nitrogen in the polycation and P is the molar amount of phosphate in the DNA). GBPEI-SS4x and GBPEI-SS8x condensed pDNA completely at N/P ratio of 5, but unmodified BPEI1.2K showed complete complexation at an N/P ratio of 10 (Figure 2). These results imply that the ability of the polymers to condensation pDNA increased as the MW of the polymers increased due to crosslinking by disulfide bonds. However, the guanidinylation ratio did not seem to affect the ability of the polymers to condense pDNA, possibly because the net positive charge of the polymers was not changed during conversion of primary amines into guanidine groups. ChemMedChem 2014, 9, 2718 – 2724
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Figure 2. Agarose gel retardation assay of pDNA complexed with a) BPEI1.2K, b) GBPEI-SS4x and c) GBPEI-SS8x at various N/P ratios. Polyplexes with BPEI1.2K, GBPEI-SS4x and GBPEI-SS8x were electrophoresed through a 1 % (w/v) agarose gel and then analyzed with a UV illuminator.
Size and Zeta-potential of polymer/pDNA polyplexes The size and surface charge of polyplexes are considered to have critical influences on the degree to which polyplexes are internalized into cells.[13] To quantify polyplex sizes, polyplexes formed with various N/P ratios were analyzed using dynamic light scattering (DLS). The particle size of the polyplex decreased as N/P ratio increased (Figure 3 a). At N/P < 5, the polyplex diameter was > 500 nm due to incomplete condensation of pDNA with polymers. However, at N/P > 5, polymers effectively condensed pDNA into nano-sized particles with a diameter approximately 200 nm. The Zeta potential of the polyplex was measured to quantify the surface charges. A positively charged surface of a polyplex enables effective adhesion to the negatively charged cell plasma membrane, and thereby increases the chance that the polyplex will be internalized into the cell.[14] The Zeta potential increased as N/P ratio increased (Figure 3 a). At N/P < 5, the zeta potential was negative (~ 25 mV), while at N/P > 5 it was positive (~ + 20 mV). These measurements of size and Zeta potential confirmed that positively charged nano-sized polyplexes formed at N/P > 5, whereas incomplete polyplexes formed at an N/P ratio of 1. The guanidinylation ratio did not seem to affect the formation of complexes. These results agree well with the results of the gel retardation assay. Cytotoxicity The cytotoxicity of GBPEI-SS was evaluated in MTT assays using human breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cell lines. Polyplexes formed by BPEI1.2K, BPEI25K, and BPEI-SS at an N/P ratio of 10 were used as controls. The BPEI1.2K/pDNA complex showed no significant cytotoxicity, but BPEI25K/pDNA complex showed severe toxicity in all cell lines; bioreducible BPEI-SS/pDNA complex showed negligible toxicity in all cell lines (Figure 4). Regardless of the guanidinyla 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. a) Size and b) Zeta potential of the BPEI-SS (&), GBPEI-SS4x (*) and GBPEI-SS8x (~) polyplex at various N/P ratios. Data represent the mean standard deviation of experiments performed in triplicate.
tion ratio, the viability of cells treated with GBPEI-SS was greater than 80 % at all N/P ratios in all cell lines tested. Because of the disulfide cleavage by glutathione in cytosol, crosslinked bioreducible BPEI can be degraded to low MW BPEI1.2K. Guanidinylation of BPEI can decrease the cytotoxicity of BPEI because the guanidine group delocalizes the strong positive charge of the primary amine in BPEI.[6, 15] In vitro transfection efficiency We further investigated in vitro gene transfection potential of the GBPEI-SS in MCF-7, MDA-MB-231, and HeLa cell lines treated with GBPEI-SS/pCMV-Luc reporter gene complex at various N/P ratios. The transfection efficiencies of each cell line treated with pDNA, BPEI25K, BPEI1.2K, or BPEI-SS were used as controls. pDNA and BPEI1.2K showed negligible transfection efficiency, whereas BPEI25K demonstrated the highest transfection efficiency in all cell lines (Figure 5). As the N/P ratio increased, the transfection efficiency of BPEI-SS increased; the maximum value occurred at an N/P ratio of 10. The enhanced transfection efficiency of reducible BPEI-SS compared with nonreducible BPEI might be due to increased pDNA release triggered by ChemMedChem 2014, 9, 2718 – 2724
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Figure 4. Cell viability profiles of BPEI1.2K, BPEI25K and BPEI-SS at N/P ratio of 10 as controls and GBPEI-SS4x, and GBPEI-SS8x at various N/P ratios in a) MCF-7, b) MDA-MB-231, and c) HeLa cell lines. Data represent the mean standard deviation of experiments performed in triplicate.
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Figure 5. Transfection efficiency of the pDNA, BPEI1.2K, and BPEI25K at N/P ratio of 10 as controls and BPEI-SS, GBPEI-SS4x and GBPEI-SS8x at various N/P ratios in a) MCF-7, b) MDA-MB-231, and c) HeLa cell lines. Data represent the mean standard deviation of experiments performed in triplicate (**p < 0.01, ***p < 0.001 determined by Student’s t-test).
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intracellular glutathione.[6e] Similarly, the transfection efficiency of GBPEI-SS increased with N/P ratio in MDA-MB-231 and HeLa cell lines, and showed a similar tendency to that of BPEI-SS in the MCF-7 cell line. Compared with BPEI-SS, which has no guanidine groups, higher transfection efficiency was observed in all cell lines treated with GBPEI-SS at all N/P ratios. This result suggests that the high gene transfection is due to the increased interaction between the cell surface and GBPEI-SS/ gene polyplex due to bidentate hydrogen bonding between the guanidine group and the phosphate of the phospholipid.[6] Investigation of cellular uptake We hypothesized that the guanidine groups on the surface of the polyplex might enhance trafficking of the polyplex into the cell. To confirm the interaction between cells and polyplex, we conducted fluorescence-activated cell sorting (FACS) analysis. Fluorescein isothiocyanate (FITC)-labeled polyplexes formed by FITC-labeled BPEI1.2K, BPEI-SS, GBPEI-SS4x and GBPEI-SS8x at an N/P ratio of 10 were transfected into MCF-7, MDA-MB-231, and HeLa cell lines. Untreated cells were used as controls. We calculated the cellular uptake efficiency (%) by regarding the M1 region as a control. BPEI1.2K showed negligible cellular uptake efficiency for all cell lines cells, whereas BPEI-SS showed enhanced cellular uptake efficiency for all cell lines, and GBPEI-SS demonstrated enhanced cellular uptake efficiency than BPEI-SS in all cell lines (Figure 6). The high degree of guanidinylation in GBPEI-SS results in high cellular uptake, possibly due to increased interaction between polyplex and cell surfaces. This result demonstrates the enhanced transfection efficiency of GBPEI-SS compared with BPEI-SS. Intracellular trafficking of polyplex To monitor intracellular trafficking of the polyplexes, we performed confocal laser scanning microscopy of the HeLa cells (Figure 7). Cells were treated with polyplex, in which polymers were labelled with FITC and pDNA was labelled with YOYO iodide. Cells treated with BPEI1.2K showed no significant fluorescence, meaning the low cellular uptake of BPEI1.2K. BPEI-SStreated cells showed yellow dots caused by the overlapped fluorescence signals of polymer and pDNA. Cells treated with GBPEI-SS4x with low degree of guanidinylation showed fluorescence signals that were similar to those of cells treated with BPEI-SS, whereas cells treated with GBPEI-SS8x with high degree of guanidinylation demonstrated much enhanced signals of polyplexes both in cytosol and in the nuclei. These results correlate well with the results of the flow cytometry study.
Conclusions We have developed guanidinylated bioreducible branched polyethylenimine (GBPEI-SS) as an efficient gene delivery carrier. GBPEI-SS can form a nano-sized complex with plasmid DNA (pDNA), and this polyplex showed appropriate physicochemical properties for gene delivery. Compared with the nonguani 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. Flow cytometry histrograms of a) MCF-7, b) MDA-MB-231, and c) HeLa cells incubated with BPEI1.2K (blue), BPEI-SS (red), GBPEI-SS4x (green) and GBPEI-SS8x (yellow) polyplex at N/P ratio of 10. Untreated cells (black) were used as controls.
dinylated system, BPEI-SS, GBPEI-SS showed enhanced transfection efficiency and intracellular uptake. Transfection efficiency increased with the number of guanidine groups in the polymer. Guanidine groups on the polyplex surface enhance celluChemMedChem 2014, 9, 2718 – 2724
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www.chemmedchem.org A calculated amount of guanidinylation agent (4 and 8 molar excess to BPEI1.2K) was added. Reaction mixtures were stirred at RT for 24 h. The product was purified by dialysis (MWCO = 100– 500 Da) against DI water for 2 d, then lyophilized. The degree of guanidinylation was calculated by using elemental analysis to compare the ratio of carbon to nitrogen (C/N) in GBPEI and BPEI. Thiolated GBPEI (GBPEI-SH) was synthesized using the previously reported method with some modification.[6d,e] GBPEI (0.767 mmol, 1 g) was dissolved in DI water, and the pH of the solution was adjusted to 7.2 by adding 1 m aq HCl. The solution was lyophilized for 2 d; this process yielded GBPEI as a yellow solid, which was dissolved in MeOH (20 mL). The resultant solution was purged with nitrogen for 15 min. Propylene sulfide (7 equiv) was added using a syringe, then the reaction mixture was stirred at 60 8C for 24 h. The product was purified by precipitation in cold Et2O (2 200 mL), and the degree of thiolation was measured using 1H NMR. To synthesize GBPEI-SS (Scheme 1), GBPEI-SH was dissolved in DMSO, and oxidative crosslinking was performed by stirring the solution at RT for 48 h. Finally, the product was purified by dialysis (MWCO = 10 kDa) against DI water and lyophilized.
Preparation of GBPEI-SS/pDNA polyplexes Figure 7. Confocal fluorescence microscopic images of a HeLa cells treated with polyplex from a) BPEI1.2K, b) BPEI-SS, c) GBPEI-SS4x, and d) GBPEI-SS8x at an N/P ratio of 10. Nuclei were stained with DAPI (blue), polymers were stained with FITC (green), and pDNA were labeled with TOTO-3 (red).
lar uptake efficiency, and the disulfide crosslinked system provides efficient release of pDNA and low cytotoxicity.
Polyplexes (polymer/pDNA complex) with 1 N/P 15, where N is the molar amount of nitrogen in the polycation and P is the molar amount of phosphate in the DNA, were prepared by adding the polymer solution to the pDNA solution. The polyplexes were incubated at RT for 30 min. The amount of DNA (20 mg mL 1) was kept constant.
Agarose gel retardation assay
Experimental Section Materials Branched polyethylenimine (BPEI) with a molecular weight (MW) of 25 kDa (BPEI25K), propylene sulfide, and 2-ethyl-2-thiopseudourea hydrobromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). BPEI with a MW of 1.2 kDa (BPEI1.2K) was obtained from Polyscience, Inc. (Warrington, PA, USA). Dialysis membranes with molecular weight cut-offs (MWCOs) of 100–500 Da and 10 kDa were purchased from Spectrum Laboratories (Rancho Domingues, CA, USA). Dulbecco’s modified Eagles’ medium (DMEM), RPMI 1640, penicillin–streptomycin, fetal bovine serum (FBS), and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Corning (Manassas, VA, USA). A luciferase assay system with reporter lysis buffer was obtained from Promega (Madison, WI, USA). Bradford protein assay reagent was obtained from Pierce Chemical Co. (Rockford, IL, USA). Plasmid DNA (pDNA) was propagated in a chemically competent DH5a strain (GibcoBRL) and prepared from overnight bacterial cultures by alkaline lysis and column purification with a Quagen plasmid Maxi kit (Valencia, CA, USA). The concentration of pDNA solution was determined by measuring the absorbance at 260 nm; its optical density from 260 to 280 nm was 1.8–1.9. Cell viability was estimated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT).
Polyplexes were loaded onto 1 % (w/v) agarose gel containing ethidium bromide (0.5 mg mL 1) with a 6 loading dye. Electrophoresis was conducted at a constant voltage of 100 V for 20 min in 0.5 Tris-aceteate-EDTA buffer. The bands were visualized under a UV illuminator to determine the location of the pDNA.
Size and Zeta potential measurements Polyplexes were prepared at various N/P ratios and diluted using DPBS (pH 7.4, 140 mm NaCl). The final pDNA concentration was adjusted to 33 mg mL 1. The mixtures were then incubated for 30 min at RT. The particle size and Zeta potential of each sample were measured by using a Zetasizer Nano S90 and Z (Malvern Instruments, Malvern, U.K.), respectively.
Cell culture Human breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cell lines were cultured in RPMI and DMEM/RPMI medium, respectively, both containing 10 % FBS and 1 % antibiotics in a humidified atmosphere with 5 % CO2 at 37 8C.
Luciferase reporter gene assay Synthesis of bioreducible guanidinylated BPEI (GBPEI-SS) Guanidinylated BPEI (GBPEI) was synthesized by following reported methods with some modification.[12] Briefly, BPEI1.2K (1.667 mmol, 2 g) in 20 mL vials was diluted to 10 mL using deionized (DI) water. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Cells were seeded on 24-well culture plates at an initial density of 6 104 cells/well, and the plates were incubated for 24 h in media (500 mL) containing 10 % FBS at 37 8C in a humidified atmosphere with 5 % CO2. To form polyplexes, a solution of pDNA (1 mg) in PBS buffer (10 mL) was added to the polymer solution (10 mL) at predeChemMedChem 2014, 9, 2718 – 2724
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CHEMMEDCHEM FULL PAPERS termined N/P ratio and incubated for 30 min at RT. The cells were then incubated with the polyplex in serum-free media (250 mL) for 4 h, then incubated for 20 h in media (500 mL) containing 10 % FBS. Cells were washed with DPBS (2 500 mL) and lysed by addition of lysis buffer (200 mL/well). Luciferase gene expression was evaluated using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer–Wellesley, MA, USA). The results are presented as mean standard deviation (n = 3).
www.chemmedchem.org Acknowledgements This work was supported by a grant of the Korea Health Technology R&D Project from the Ministry of Health & Welfare, Republic of Korea (A111803). Keywords: cell-penetrating peptides · gene delivery · gene expression · guanidine · polycations · redox chemistry
MTT assay The cytotoxicity of GBPEI-SS/pDNA complexes was evaluated using a standard MTT assay protocol. Briefly, cells were seeded in 96-well plates at a density of 5 103 cells/well, and the plates were incubated for 24 h in a humidified atmosphere with 5 % CO2 at 37 8C. pDNA (0.4 mg mL 1) was complexed with the polymer at predetermined N/P ratios in DPBS and incubated for 30 min before use. Polyplexes were incubated for 4 h with the cells in serum-free media (100 mL), then for 20 h in fresh media (200 mL) containing 10 % FBS. These mixtures were mixed with MTT solution (20 mL, 5 mg mL 1) and incubated for another 4 h. The media was removed, and DMSO (150 mL) was added to each well to dissolve the internalized purple formazan crystals. An aliquot of 100 mL was taken from each well and transferred to a well of a fresh 96-well plate. The absorption was measured at 570 nm using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer– Wellesley, MA, USA). The relative percentage of the control (untreated) cells, which were not exposed to the transfection system, was used to represent 100 % cell viability.
Fluorescence-activated cell sorting (FACS) assay Cells were seeded into 12-well plates at a density of 1 105 cells/ well, and the plates were incubated for 24 h in a humidified atmosphere with 5 % CO2 at 37 8C. pDNA (1.0 mg mL 1) was complexed with polymer at an N/P ratio of 10 in DPBS and incubated for 30 min before use. Polyplexes were incubated with the cells for 4 h in serum-free medium (500 mL). After incubation, the cells were washed with DPBS (2 1 mL), then trypsinized. The harvested cells were centrifuged and supernatant was removed, then the cells were fixed with 4 % paraformaldehyde solution at 4 8C overnight. Paraformaldehyde solution was removed by centrifugation, and then the cells were resuspended in DPBS. The cells were analyzed using a FACS Calibur (Becton Dickinson, San Jose, CA, USA) and Becton Dickinson Cell Quest software, following the manufacturers’ instructions.
Confocal laser scanning microscopy Cells were seeded into 12-well plates over glass coverslips at a density of 1 105 cells/well and incubated for 24 h. pDNA (1.0 mg mL 1) was complexed with the polymer at an N/P ratio of 10 in DPBS and incubated for 30 min before use. Polyplexes were incubated with the cells for 4 h in serum-free media (500 mL). After incubation, the cells were washed with DPBS (2 1 mL) then fixed with 4 % paraformaldehyde solution at 4 8C overnight. Cells on the coverslip were mounted in Vectashield antifade mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vetor Labs), and then the cells were observed using a confocal laser scanning microscope and analyzed using OLYMPUS FLUOVIEW version 1.5 Viewer software.
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[1] a) S. De Smedt, J. Demeester, W. Hennink, Pharm. Res. 2000, 17, 113 – 126; b) M. R. Dyer, P. L. Herrling, Mol. Ther. 2000, 1, 213 – 224; c) S. Son, R. Namgung, J. Kim, K. Singha, W. J. Kim, Acc. Chem. Res. 2012, 45, 1100 – 1112. [2] a) O. Boussif, F. Lezoualc’h, M. A. Zanta, M. D. Mergny, D. Scherman, B. Demeneix, J. P. Behr, Proc. Natl. Acad. Sci. USA 1995, 92, 7297 – 7301; b) H. Kim, R. Namgung, K. Singha, I.-K. Oh, W. J. Kim, Bioconjugate Chem. 2011, 22, 2558 – 2567; c) R. Namgung, W. J. Kim, Small 2012, 8, 3209 – 3219; d) N. S. Templeton, D. D. Lasic, P. M. Frederik, H. H. Strey, D. D. Roberts, G. N. Pavlakis, Nat. Biotechnol. 1997, 15, 647 – 652; e) S. Herlambang, M. Kumagai, T. Nomoto, S. Horie, S. Fukushima, M. Oba, K. Miyazaki, Y. Morimoto, N. Nishiyama, K. Kataoka, J. Controlled Release 2011, 155, 449 – 457. [3] S. Dinc¸er, M. Trk, E. Piskin, Gene Ther. 2005, 12, S139 – S145. [4] D. W. Pack, A. S. Hoffman, S. Pun, P. S. Stayton, Nat. Rev. Drug Discovery 2005, 4, 581 – 593. [5] a) W. T. Godbey, K. K. Wu, A. G. Mikos, J. Biomed. Mater. Res. 1999, 45, 268 – 275; b) D. Fischer, T. Bieber, Y. Li, H.-P. Elssser, T. Kissel, Pharm. Res. 1999, 16, 1273 – 1279. [6] a) S. Choi, K.-D. Lee, J. Controlled Release 2008, 131, 70 – 76; b) M. A. Gosselin, W. Guo, R. J. Lee, Bioconjugate Chem. 2001, 12, 989 – 994; c) D. W. Hwang, S. Son, J. Jang, H. Youn, S. Lee, D. Lee, Y.-S. Lee, J. M. Jeong, W. J. Kim, D. S. Lee, Biomaterials 2011, 32, 4968 – 4975; d) Q. Peng, Z. Zhong, R. Zhuo, Bioconjugate Chem. 2008, 19, 499 – 506; e) S. Son, K. Singha, W. J. Kim, Biomaterials 2010, 31, 6344 – 6354; f) F. Meng, W. E. Hennink, Z. Zhong, Biomaterials 2009, 30, 2180 – 2198. [7] A. Meister, M. E. Anderson, Annu. Rev. Biochem. 1983, 52, 711 – 760. [8] a) F. Milletti, Drug Discovery Today 2012, 17, 850 – 860; b) L. Patel, J. Zaro, W.-C. Shen, Pharm. Res. 2007, 24, 1977 – 1992; c) M. Zorko, . Langel, Adv. Drug Delivery Rev. 2005, 57, 529 – 545. [9] a) P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, J. B. Rothbard, Proc. Natl. Acad. Sci. USA 2000, 97, 13003 – 13008; b) E. Koren, V. P. Torchilin, Trends Mol. Med. 2012, 18, 385 – 393. [10] T. A. Theodossiou, A. Pantos, I. Tsogas, C. M. Paleos, ChemMedChem 2008, 3, 1635 – 1643. [11] a) L. Bromberg, S. Raduyk, T. A. Hatton, A. Concheiro, C. Rodriguez-Valencia, M. Silva, C. Alvarez-Lorenzo, Bioconjugate Chem. 2009, 20, 1044 – 1053; b) A. M. Funhoff, C. F. van Nostrum, M. C. Lok, M. M. Fretz, D. J. A. Crommelin, W. E. Hennink, Bioconjugate Chem. 2004, 15, 1212 – 1220; c) T.-i. Kim, M. Lee, S. W. Kim, Biomaterials 2010, 31, 1798 – 1804; d) T.-i. Kim, T. Rothmund, T. Kissel, S. W. Kim, J. Controlled Release 2011, 152, 110 – 119; e) S. Nimesh, R. Chandra, Eur. J. Pharm. Biopharm. 2008, 68, 647 – 655; f) J.-H. Yu, J. Huang, H.-L. Jiang, J.-S. Quan, M.-H. Cho, C.-S. Cho, J. Appl. Polym. Sci. 2009, 112, 926 – 933. [12] L. Fishbein, J. A. Gallaghan, J. Am. Chem. Soc. 1954, 76, 3217 – 3219. [13] J.-Y. Cherng, P. van de Wetering, H. Talsma, D. A. Crommelin, W. Hennink, Pharm. Res. 1996, 13, 1038 – 1042. [14] I. Kopatz, J.-S. Remy, J.-P. Behr, J. Gene Med. 2004, 6, 769 – 776. [15] N. Cheng, W. Liu, Z. Cao, W. Ji, D. Liang, G. Guo, J. Zhang, Biomaterials 2006, 27, 4984 – 4992. Received: July 16, 2014 Published online on October 6, 2014
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Bioreducible Guanidinylated Polyethylenimine for Efficient Gene Delivery Duhwan Lee,[a] Yeong Mi Lee,[a] Cherlhyun Jeong,[b] Jun Lee,[c] and Won Jong Kim*[a] Cationic polymers are known to afford efficient gene transfection. However, cytotoxicity remains a problem at the molecular weight for optimal DNA delivery. As such, optimized polymeric gene delivery systems are still a sought-after research goal. A guanidinylated bioreducible branched polyethylenimine (GBPEI-SS) was synthesized by using a disulfide bond to crosslink the guanidinylated BPEI (GBPEI). GBPEI-SS showed sufficient plasmid DNA (pDNA) condensation ability. The physicochemical properties of GBPEI-SS demonstrate that it has the appropriate size (~ 200 nm) and surface potential (~ 30 mV) at
a nitrogen-to-phosphorus ratio of 10. No significant toxicity was observed, possibly due to bioreducibility and to the guanidine group delocalizing the positive charge of the primary amine in BPEI. Compared with the nonguanidinylated analogue, BPEI-SS, GBPEI-SS showed enhanced transfection efficiency owing to increased cellular uptake and efficient pDNA release by cleavage of disulfide bonds. This system is very efficient for delivering pDNA into cells, thereby achieving high transfection efficiency and low cytotoxicity.
Introduction Nonviral gene delivery systems can overcome the demerits of viral gene delivery systems, such as the host immune response, low capacity for genes, risk of replication, and random DNA insertion.[1] Several nonviral gene delivery systems use cationic polymers, liposomes, self-assembled nanoparticles or inorganic nanoparticles.[2] These nonviral vectors have advantages of facile synthesis and modification, non-immunogenicity, large capacity for genes, and low cytotoxicity.[3] Polymeric gene delivery systems are suitable candidates for efficient gene delivery due to the spontaneous formation of a nano-sized complex (polyplex) with negatively charged DNA by electrostatic interaction, the efficient internalization into the cell, and the easy endosomal escape.[4] Branched and linear polyethylenimine (PEI), chitosan, poly-llysine, and many cationic polymers have been developed to deliver DNA to cells. Branched PEI (BPEI) is one of the most effective cationic polymers and is regarded as the standard to [a] D. Lee,+ Dr. Y. M. Lee,+ Prof. Dr. W. J. Kim Center for Self-assembly and Complexity, Institute for Basic Science and Department of Chemistry Pohang University of Science and Technology (POSTECH) San 31, Hyoja-dong, Pohang 790-784 (Republic of Korea) E-mail: [email protected] [b] Dr. C. Jeong Center for Theragnosis, Biomedical Research Institute Korea Institute of Science and Technology (KIST) Hwarang-ro 14-gil 5, Seongbuk-gu, Seoul 136-791 (Republic of Korea) [c] Dr. J. Lee Department of Oral & Maxillofacial Surgery, School of dentistry Wonkwang Bone Regeneration Research Institute Wonkwang University, Iksan 570-749 (Republic of Korea) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201402293.
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which polymeric gene delivery systems should be compared. However, the transfection efficiency of BPEI depends on its molecular weight (MW) along with its nonspecific cytotoxicity. BPEI with high MW (> 25 kDa) shows high transfection efficiency with significant cytotoxicity, whereas BPEI with low MW (< 1.8 kDa) shows low transfection efficiency with low cytotoxicity.[5] To obtain high transfection efficiency and low cytotoxicity, researchers have introduced the strategy of using bioreducible disulfide linkage to crosslink low MW BPEI.[6] In the extracellular compartments, this bioreducible polymer forms stable polyplexes due to low concentrations of reducing agents such as glutathione. However, in intracellular compartments, disulfide bonds can be reduced and cleaved to thiol groups due to the reducing environment of cytosolic compartments. As a result, bioreducible crosslinked BPEI can be degraded to low MW BPEI, which releases its DNA contents efficiently.[6d, 7] However, to increase gene transfection efficiency, the cellular uptake of crosslinked BPEI into cells remains to be optimized. To achieve this goal, we were inspired by the functionality of cell-penetrating peptides (CPPs). CPPs, short peptides of 30 or fewer amino acids, facilitate uptake of various molecular cargos into cells. Therefore, conjugation of the carriers with CPPs such as HIV-1 Tat peptide and Drosophila antennapedia homoprotein can enhance cellular uptake, and thus increase the delivery efficiency. CPPs have cationic or amphiphilic amino acid moieties, such as arginine and lysine residues, in their sequence.[8] Arginine residues of CPPs contribute strongly to their high internalization into cells.[9] The guanidine functional group of the arginine residue can interact with phosphate, sulfate or carboxylate of the proteoglycans on the cell surface by bidentate hydrogen bonding.[10] Inspired by this strategy, researchers have reported guanidinylated polymeric gene delivery systems that use poly(allylChemMedChem 2014, 9, 2718 – 2724
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CHEMMEDCHEM FULL PAPERS amine), polyethylenimine–polyoxypropylene–polyoxyethylene, cationic polymethacrylate, or poly(cystaminebisacrylamide-diaminohexane). These guanidinylated polymers show high gene transfection efficiency and improved intracellular uptake over nonguanidinylated analogues.[11] Therefore, we expect that conjugation of a guanidine group to bioreducible polymers will increase their gene delivery efficiency by increasing cellular uptake and the efficiency of gene release. Moreover, conjugation of the guanidine group to a BPEI can be expected to decrease its cytotoxicity by converting the primary amine to a guanidine group (Figure 1). In this study, guanidinylated disulfide crosslinked BPEI (GBPEI-SS) was synthesized, and the physicochemical properties of the polyplexes were investigated. Transfection efficiency, cytotoxicity and cellular uptake efficiency were also evaluated.
Figure 1. Schematic diagram of gene delivery based on the bioreducible guanidinylated branched polyethylenimine (GBPEI-SS) polyplex.
Results and Discussion
www.chemmedchem.org Table 1. Characterization of nonguanidinylated (BPEI-SS) and guanidinylated (GBPEI-SS) bioreducible branched polyethylenimine. Denotation
BPEI-SS GBPEI-SS4x GBPEI-SS8x
Thiol group Feed ratio[a] Degree of thiolation[b] 7 7 7
6.55 5.42 4.58
Guanidine group Feed ratio[c] Degree of guanidinylation[d] – 4 8
– 2.4 (34.4) 4.3 (61.6)
[a] Propylene sulfide/BPEI1.2K molar ratio. [b] Total quantities of thiol in one chain of BPEI1.2K, measured by 1H NMR. [c] 2-Ethyl-2-thiopsuedourea hydrobromide/BPEI1.2K molar ratio. [d] Total quantity of guanidine in one chain of BPEI1.2K, measured by elemental analysis. Values in parentheses are the percentage of primary amines converted into guanidine group.
termined by comparing the C/N ratio between BPEI1.2K and GBPEI. Using four and eight equivalents of 2-ethyl-2-thiopseudourea gave a conjugation ratio of 2.4 (GBPEI4x) and 4.3 (GBPEI8x), respectively (Table 1). GBPEI was endowed with bioreducibility by using a disulfide bond to crosslink it. Using propylene sulfide as a thiolation reagent (7 equiv to BPEI1.2K), thiol groups were conjugated to primary and secondary amine groups of GBPEI. During the reaction, the buffering capacity of GBPEI did not change significantly because primary amine and secondary amine were transformed to higher order amine groups.[6d] The degree of thiolation was determined using 1H NMR. The calculated conjugation ratios were 5.42 and 4.58 for GBPEI-SS4x and GBPEI-SS8x, respectively. As a control, bioreducible BPEI with no guanidine group (BPEI-SS) was also synthesized. The degree of thiolation of BPEI-SS was 6.55. The successful synthesis of GBPEI-SS was also confirmed using 1H NMR. In the 1H NMR spectra, a methyl proton adjacent to thiol (d = 1.2–1.5 ppm), methylene proton of BPEI (d = 2.5– 3.2 ppm) and methylene proton adjacent to guanidine group (d = 3.2–3.8 ppm) appeared.
Synthesis and characterization of GBPEI-SS For the synthesis of GBPEI-SS (Scheme 1), a guanidine group was first introduced to low MW BPEI1.2K. To achieve this, 2ethyl-2-thiopseudourea hydrobromide was used as a guanidinylation agent at different molar ratios (4 and 8 equiv). During the reaction, ethanethiol was generated as a byproduct through attack of the thiopseudourea carbon by the primary amine group of BPEI. After purification by dialysis, elemental analysis was used to confirm that a guanidine group had been introduced to BPEI. The conjugation ratio of guanidine was de-
Scheme 1. Preparation of bioreducible guanidinylated branched polyethylenimine (GBPEI-SS). Reagents and conditions: a) MeOH, RT, 24 h; b) 1. propylene sulfide, MeOH, 60 8C, 24 h; 2. DMSO, RT, 48 h.
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Physicochemical property of polyplexes For efficient gene delivery, the cationic polymer that forms the polyplex with plasmid DNA (pDNA) should condense pDNA effectively. An agarose gel retardation assay was conducted to investigate the formation of the GBPEI-SS/pDNA complex at different N/P ratios (where N is the molar amount of nitrogen in the polycation and P is the molar amount of phosphate in the DNA). GBPEI-SS4x and GBPEI-SS8x condensed pDNA completely at N/P ratio of 5, but unmodified BPEI1.2K showed complete complexation at an N/P ratio of 10 (Figure 2). These results imply that the ability of the polymers to condensation pDNA increased as the MW of the polymers increased due to crosslinking by disulfide bonds. However, the guanidinylation ratio did not seem to affect the ability of the polymers to condense pDNA, possibly because the net positive charge of the polymers was not changed during conversion of primary amines into guanidine groups. ChemMedChem 2014, 9, 2718 – 2724
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Figure 2. Agarose gel retardation assay of pDNA complexed with a) BPEI1.2K, b) GBPEI-SS4x and c) GBPEI-SS8x at various N/P ratios. Polyplexes with BPEI1.2K, GBPEI-SS4x and GBPEI-SS8x were electrophoresed through a 1 % (w/v) agarose gel and then analyzed with a UV illuminator.
Size and Zeta-potential of polymer/pDNA polyplexes The size and surface charge of polyplexes are considered to have critical influences on the degree to which polyplexes are internalized into cells.[13] To quantify polyplex sizes, polyplexes formed with various N/P ratios were analyzed using dynamic light scattering (DLS). The particle size of the polyplex decreased as N/P ratio increased (Figure 3 a). At N/P < 5, the polyplex diameter was > 500 nm due to incomplete condensation of pDNA with polymers. However, at N/P > 5, polymers effectively condensed pDNA into nano-sized particles with a diameter approximately 200 nm. The Zeta potential of the polyplex was measured to quantify the surface charges. A positively charged surface of a polyplex enables effective adhesion to the negatively charged cell plasma membrane, and thereby increases the chance that the polyplex will be internalized into the cell.[14] The Zeta potential increased as N/P ratio increased (Figure 3 a). At N/P < 5, the zeta potential was negative (~ 25 mV), while at N/P > 5 it was positive (~ + 20 mV). These measurements of size and Zeta potential confirmed that positively charged nano-sized polyplexes formed at N/P > 5, whereas incomplete polyplexes formed at an N/P ratio of 1. The guanidinylation ratio did not seem to affect the formation of complexes. These results agree well with the results of the gel retardation assay. Cytotoxicity The cytotoxicity of GBPEI-SS was evaluated in MTT assays using human breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cell lines. Polyplexes formed by BPEI1.2K, BPEI25K, and BPEI-SS at an N/P ratio of 10 were used as controls. The BPEI1.2K/pDNA complex showed no significant cytotoxicity, but BPEI25K/pDNA complex showed severe toxicity in all cell lines; bioreducible BPEI-SS/pDNA complex showed negligible toxicity in all cell lines (Figure 4). Regardless of the guanidinyla 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. a) Size and b) Zeta potential of the BPEI-SS (&), GBPEI-SS4x (*) and GBPEI-SS8x (~) polyplex at various N/P ratios. Data represent the mean standard deviation of experiments performed in triplicate.
tion ratio, the viability of cells treated with GBPEI-SS was greater than 80 % at all N/P ratios in all cell lines tested. Because of the disulfide cleavage by glutathione in cytosol, crosslinked bioreducible BPEI can be degraded to low MW BPEI1.2K. Guanidinylation of BPEI can decrease the cytotoxicity of BPEI because the guanidine group delocalizes the strong positive charge of the primary amine in BPEI.[6, 15] In vitro transfection efficiency We further investigated in vitro gene transfection potential of the GBPEI-SS in MCF-7, MDA-MB-231, and HeLa cell lines treated with GBPEI-SS/pCMV-Luc reporter gene complex at various N/P ratios. The transfection efficiencies of each cell line treated with pDNA, BPEI25K, BPEI1.2K, or BPEI-SS were used as controls. pDNA and BPEI1.2K showed negligible transfection efficiency, whereas BPEI25K demonstrated the highest transfection efficiency in all cell lines (Figure 5). As the N/P ratio increased, the transfection efficiency of BPEI-SS increased; the maximum value occurred at an N/P ratio of 10. The enhanced transfection efficiency of reducible BPEI-SS compared with nonreducible BPEI might be due to increased pDNA release triggered by ChemMedChem 2014, 9, 2718 – 2724
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Figure 4. Cell viability profiles of BPEI1.2K, BPEI25K and BPEI-SS at N/P ratio of 10 as controls and GBPEI-SS4x, and GBPEI-SS8x at various N/P ratios in a) MCF-7, b) MDA-MB-231, and c) HeLa cell lines. Data represent the mean standard deviation of experiments performed in triplicate.
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Figure 5. Transfection efficiency of the pDNA, BPEI1.2K, and BPEI25K at N/P ratio of 10 as controls and BPEI-SS, GBPEI-SS4x and GBPEI-SS8x at various N/P ratios in a) MCF-7, b) MDA-MB-231, and c) HeLa cell lines. Data represent the mean standard deviation of experiments performed in triplicate (**p < 0.01, ***p < 0.001 determined by Student’s t-test).
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intracellular glutathione.[6e] Similarly, the transfection efficiency of GBPEI-SS increased with N/P ratio in MDA-MB-231 and HeLa cell lines, and showed a similar tendency to that of BPEI-SS in the MCF-7 cell line. Compared with BPEI-SS, which has no guanidine groups, higher transfection efficiency was observed in all cell lines treated with GBPEI-SS at all N/P ratios. This result suggests that the high gene transfection is due to the increased interaction between the cell surface and GBPEI-SS/ gene polyplex due to bidentate hydrogen bonding between the guanidine group and the phosphate of the phospholipid.[6] Investigation of cellular uptake We hypothesized that the guanidine groups on the surface of the polyplex might enhance trafficking of the polyplex into the cell. To confirm the interaction between cells and polyplex, we conducted fluorescence-activated cell sorting (FACS) analysis. Fluorescein isothiocyanate (FITC)-labeled polyplexes formed by FITC-labeled BPEI1.2K, BPEI-SS, GBPEI-SS4x and GBPEI-SS8x at an N/P ratio of 10 were transfected into MCF-7, MDA-MB-231, and HeLa cell lines. Untreated cells were used as controls. We calculated the cellular uptake efficiency (%) by regarding the M1 region as a control. BPEI1.2K showed negligible cellular uptake efficiency for all cell lines cells, whereas BPEI-SS showed enhanced cellular uptake efficiency for all cell lines, and GBPEI-SS demonstrated enhanced cellular uptake efficiency than BPEI-SS in all cell lines (Figure 6). The high degree of guanidinylation in GBPEI-SS results in high cellular uptake, possibly due to increased interaction between polyplex and cell surfaces. This result demonstrates the enhanced transfection efficiency of GBPEI-SS compared with BPEI-SS. Intracellular trafficking of polyplex To monitor intracellular trafficking of the polyplexes, we performed confocal laser scanning microscopy of the HeLa cells (Figure 7). Cells were treated with polyplex, in which polymers were labelled with FITC and pDNA was labelled with YOYO iodide. Cells treated with BPEI1.2K showed no significant fluorescence, meaning the low cellular uptake of BPEI1.2K. BPEI-SStreated cells showed yellow dots caused by the overlapped fluorescence signals of polymer and pDNA. Cells treated with GBPEI-SS4x with low degree of guanidinylation showed fluorescence signals that were similar to those of cells treated with BPEI-SS, whereas cells treated with GBPEI-SS8x with high degree of guanidinylation demonstrated much enhanced signals of polyplexes both in cytosol and in the nuclei. These results correlate well with the results of the flow cytometry study.
Conclusions We have developed guanidinylated bioreducible branched polyethylenimine (GBPEI-SS) as an efficient gene delivery carrier. GBPEI-SS can form a nano-sized complex with plasmid DNA (pDNA), and this polyplex showed appropriate physicochemical properties for gene delivery. Compared with the nonguani 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 6. Flow cytometry histrograms of a) MCF-7, b) MDA-MB-231, and c) HeLa cells incubated with BPEI1.2K (blue), BPEI-SS (red), GBPEI-SS4x (green) and GBPEI-SS8x (yellow) polyplex at N/P ratio of 10. Untreated cells (black) were used as controls.
dinylated system, BPEI-SS, GBPEI-SS showed enhanced transfection efficiency and intracellular uptake. Transfection efficiency increased with the number of guanidine groups in the polymer. Guanidine groups on the polyplex surface enhance celluChemMedChem 2014, 9, 2718 – 2724
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www.chemmedchem.org A calculated amount of guanidinylation agent (4 and 8 molar excess to BPEI1.2K) was added. Reaction mixtures were stirred at RT for 24 h. The product was purified by dialysis (MWCO = 100– 500 Da) against DI water for 2 d, then lyophilized. The degree of guanidinylation was calculated by using elemental analysis to compare the ratio of carbon to nitrogen (C/N) in GBPEI and BPEI. Thiolated GBPEI (GBPEI-SH) was synthesized using the previously reported method with some modification.[6d,e] GBPEI (0.767 mmol, 1 g) was dissolved in DI water, and the pH of the solution was adjusted to 7.2 by adding 1 m aq HCl. The solution was lyophilized for 2 d; this process yielded GBPEI as a yellow solid, which was dissolved in MeOH (20 mL). The resultant solution was purged with nitrogen for 15 min. Propylene sulfide (7 equiv) was added using a syringe, then the reaction mixture was stirred at 60 8C for 24 h. The product was purified by precipitation in cold Et2O (2 200 mL), and the degree of thiolation was measured using 1H NMR. To synthesize GBPEI-SS (Scheme 1), GBPEI-SH was dissolved in DMSO, and oxidative crosslinking was performed by stirring the solution at RT for 48 h. Finally, the product was purified by dialysis (MWCO = 10 kDa) against DI water and lyophilized.
Preparation of GBPEI-SS/pDNA polyplexes Figure 7. Confocal fluorescence microscopic images of a HeLa cells treated with polyplex from a) BPEI1.2K, b) BPEI-SS, c) GBPEI-SS4x, and d) GBPEI-SS8x at an N/P ratio of 10. Nuclei were stained with DAPI (blue), polymers were stained with FITC (green), and pDNA were labeled with TOTO-3 (red).
lar uptake efficiency, and the disulfide crosslinked system provides efficient release of pDNA and low cytotoxicity.
Polyplexes (polymer/pDNA complex) with 1 N/P 15, where N is the molar amount of nitrogen in the polycation and P is the molar amount of phosphate in the DNA, were prepared by adding the polymer solution to the pDNA solution. The polyplexes were incubated at RT for 30 min. The amount of DNA (20 mg mL 1) was kept constant.
Agarose gel retardation assay
Experimental Section Materials Branched polyethylenimine (BPEI) with a molecular weight (MW) of 25 kDa (BPEI25K), propylene sulfide, and 2-ethyl-2-thiopseudourea hydrobromide were purchased from Sigma–Aldrich (St. Louis, MO, USA). BPEI with a MW of 1.2 kDa (BPEI1.2K) was obtained from Polyscience, Inc. (Warrington, PA, USA). Dialysis membranes with molecular weight cut-offs (MWCOs) of 100–500 Da and 10 kDa were purchased from Spectrum Laboratories (Rancho Domingues, CA, USA). Dulbecco’s modified Eagles’ medium (DMEM), RPMI 1640, penicillin–streptomycin, fetal bovine serum (FBS), and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Corning (Manassas, VA, USA). A luciferase assay system with reporter lysis buffer was obtained from Promega (Madison, WI, USA). Bradford protein assay reagent was obtained from Pierce Chemical Co. (Rockford, IL, USA). Plasmid DNA (pDNA) was propagated in a chemically competent DH5a strain (GibcoBRL) and prepared from overnight bacterial cultures by alkaline lysis and column purification with a Quagen plasmid Maxi kit (Valencia, CA, USA). The concentration of pDNA solution was determined by measuring the absorbance at 260 nm; its optical density from 260 to 280 nm was 1.8–1.9. Cell viability was estimated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT).
Polyplexes were loaded onto 1 % (w/v) agarose gel containing ethidium bromide (0.5 mg mL 1) with a 6 loading dye. Electrophoresis was conducted at a constant voltage of 100 V for 20 min in 0.5 Tris-aceteate-EDTA buffer. The bands were visualized under a UV illuminator to determine the location of the pDNA.
Size and Zeta potential measurements Polyplexes were prepared at various N/P ratios and diluted using DPBS (pH 7.4, 140 mm NaCl). The final pDNA concentration was adjusted to 33 mg mL 1. The mixtures were then incubated for 30 min at RT. The particle size and Zeta potential of each sample were measured by using a Zetasizer Nano S90 and Z (Malvern Instruments, Malvern, U.K.), respectively.
Cell culture Human breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cell lines were cultured in RPMI and DMEM/RPMI medium, respectively, both containing 10 % FBS and 1 % antibiotics in a humidified atmosphere with 5 % CO2 at 37 8C.
Luciferase reporter gene assay Synthesis of bioreducible guanidinylated BPEI (GBPEI-SS) Guanidinylated BPEI (GBPEI) was synthesized by following reported methods with some modification.[12] Briefly, BPEI1.2K (1.667 mmol, 2 g) in 20 mL vials was diluted to 10 mL using deionized (DI) water. 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Cells were seeded on 24-well culture plates at an initial density of 6 104 cells/well, and the plates were incubated for 24 h in media (500 mL) containing 10 % FBS at 37 8C in a humidified atmosphere with 5 % CO2. To form polyplexes, a solution of pDNA (1 mg) in PBS buffer (10 mL) was added to the polymer solution (10 mL) at predeChemMedChem 2014, 9, 2718 – 2724
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CHEMMEDCHEM FULL PAPERS termined N/P ratio and incubated for 30 min at RT. The cells were then incubated with the polyplex in serum-free media (250 mL) for 4 h, then incubated for 20 h in media (500 mL) containing 10 % FBS. Cells were washed with DPBS (2 500 mL) and lysed by addition of lysis buffer (200 mL/well). Luciferase gene expression was evaluated using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer–Wellesley, MA, USA). The results are presented as mean standard deviation (n = 3).
www.chemmedchem.org Acknowledgements This work was supported by a grant of the Korea Health Technology R&D Project from the Ministry of Health & Welfare, Republic of Korea (A111803). Keywords: cell-penetrating peptides · gene delivery · gene expression · guanidine · polycations · redox chemistry
MTT assay The cytotoxicity of GBPEI-SS/pDNA complexes was evaluated using a standard MTT assay protocol. Briefly, cells were seeded in 96-well plates at a density of 5 103 cells/well, and the plates were incubated for 24 h in a humidified atmosphere with 5 % CO2 at 37 8C. pDNA (0.4 mg mL 1) was complexed with the polymer at predetermined N/P ratios in DPBS and incubated for 30 min before use. Polyplexes were incubated for 4 h with the cells in serum-free media (100 mL), then for 20 h in fresh media (200 mL) containing 10 % FBS. These mixtures were mixed with MTT solution (20 mL, 5 mg mL 1) and incubated for another 4 h. The media was removed, and DMSO (150 mL) was added to each well to dissolve the internalized purple formazan crystals. An aliquot of 100 mL was taken from each well and transferred to a well of a fresh 96-well plate. The absorption was measured at 570 nm using a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer– Wellesley, MA, USA). The relative percentage of the control (untreated) cells, which were not exposed to the transfection system, was used to represent 100 % cell viability.
Fluorescence-activated cell sorting (FACS) assay Cells were seeded into 12-well plates at a density of 1 105 cells/ well, and the plates were incubated for 24 h in a humidified atmosphere with 5 % CO2 at 37 8C. pDNA (1.0 mg mL 1) was complexed with polymer at an N/P ratio of 10 in DPBS and incubated for 30 min before use. Polyplexes were incubated with the cells for 4 h in serum-free medium (500 mL). After incubation, the cells were washed with DPBS (2 1 mL), then trypsinized. The harvested cells were centrifuged and supernatant was removed, then the cells were fixed with 4 % paraformaldehyde solution at 4 8C overnight. Paraformaldehyde solution was removed by centrifugation, and then the cells were resuspended in DPBS. The cells were analyzed using a FACS Calibur (Becton Dickinson, San Jose, CA, USA) and Becton Dickinson Cell Quest software, following the manufacturers’ instructions.
Confocal laser scanning microscopy Cells were seeded into 12-well plates over glass coverslips at a density of 1 105 cells/well and incubated for 24 h. pDNA (1.0 mg mL 1) was complexed with the polymer at an N/P ratio of 10 in DPBS and incubated for 30 min before use. Polyplexes were incubated with the cells for 4 h in serum-free media (500 mL). After incubation, the cells were washed with DPBS (2 1 mL) then fixed with 4 % paraformaldehyde solution at 4 8C overnight. Cells on the coverslip were mounted in Vectashield antifade mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vetor Labs), and then the cells were observed using a confocal laser scanning microscope and analyzed using OLYMPUS FLUOVIEW version 1.5 Viewer software.
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