Macromolecular Research, Vol. 22, No. 7, pp 757-764 (2014) DOI 10.1007/s13233-014-2108-8
www.springer.com/13233 pISSN 1598-5032 eISSN 2092-7673
Polyethylenimine-Grafted Polyamidoamine Conjugates for Gene Delivery with High Efficiency and Low Cytotoxicity Tae-Hun Kim1, Ho Won Seo2, Jin Han3, Kyung Soo Ko*,4, and Joon Sig Choi*,1,5 1
Department of Biochemistry, Chungnam National University, Daejeon 305-764, Korea 2 Graduate School of Nanoscience and Technology (GSNT), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea 3 Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan 614-735, Korea 4 Department of Internal Medicine, Sanggye Paik Hospital, Cardiovascular and Metabolic Disease Center, Inje University, Seoul 139-707, Korea 5 Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Korea Received January 22, 2014; Revised March 31, 2014; Accepted April 17, 2014 Abstract: Hyperbranched polymers, polyamidoamine-methyl acrylate-polyethylenimine-ethylenediamine (PMPE) conjugates, were synthesized as a novel non-viral gene carrier based on polyamidoamine. These PMPE derivatives exhibit high transfection efficiency and low cytotoxicity. We linked methyl acrylate (MA) on polyamidoamine (PAMAM) and grafted PEI onto the PAMAM-MA, and then grafted ethylenediamine (EDA) onto the PAMAM-MA-PEI. Four polymers, PAMAM G2-MA-PEI 800-EDA, PAMAM G3-MA-PEI 800-EDA and PAMAM G2-MA-PEI 2000-EDA, PAMAM G3-MA-PEI 2000-EDA, were synthesized and characterized by 1H NMR. PMPE was shown to interact with and condense plasmid DNA effectively to form 149-220 nm polyplexes with 34-43 mV of zeta potentials at weight ratio as 4:1 (polymer/plasmid DNA). Cytotoxicity of PMPE/pDNA complexes was lower than that of polyethylenimine (PEI) 25 kDa/pDNA complexes for all concentration ranges. In 293 and HeLa cells, PMPE/pDNA complexes showed much higher gene transfection efficiency than PAMAM. These results suggested that PMPE is an attractive novel vector for non-viral gene delivery system. Keywords: PAMAM dendrimer, polyethylenimine, polyplexes, nanoparticles, transfection.
Introduction
tion by protecting DNA from nuclease degradation and facilitating its cellular uptake and intracellular traffic into the nucleus. Among cationic polymeric non-viral vectors, dendrimer, polyamidoamine (PAMAM) was widely used in vitro and in vivo gene delivery systems.5-7 Dendrimers are core-shell macromolecules that have highly ordered structure, low polydispersity and multiple terminal functional groups. PAMAM are based on an ethylenediamine core and an amidoamine repeat branching structure. PAMAM grows linearly in diameter as addition of successive layers (generations), and amplifies exponentially the number of surface groups. PAMAM range in diameter from 1.1 nm (G1) up to 12.4 nm (G10).8 The diameters of these increases by about 1 nm per generation. Highgeneration PAMAM exhibits a high transfection efficiency but also high cytotoxicity. In contrast, low-generation PAMAM is less toxic (than the high-generation PAMAM) but also cannot be used as gene delivery vectors because of its low transfection efficiency.9-11 Therefore, many techniques have been tried to solve these problems of PAMAM, including acetylation to reduce toxicity,12 conjugation functional peptides or
Gene therapy has received significant attention in the last few decades because of its potential application in the treatment of currently incurable inheritable or acquired diseases.1-3 The main objective in gene therapy is successful in vivo transfer of the genetic materials to the targeted cells or organs. However, naked therapeutic genes are rapidly degraded by serum nucleases in the blood before they can reach the target site.4 Therefore, the present study is mainly focused on the development of safe and efficient gene carriers. Non-viral vectors for gene delivery have several advantages such as non-immunogenicity, the ability to transfer large and diverse genetic materials, and the potential for diverse modifications of their structures. The major categories of non-viral vectors include cationic polymers and cationic lipids. Both cationic lipid and cationic polymer systems are used to enhance gene transfec*Corresponding Authors. E-mails:
[email protected] or
[email protected] The Polymer Society of Korea
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amino acids to enhance transfection efficacy.13,14 Polyethylenimine (PEI) is synthesized through the acid catalyzed ring-opening polymerization of ethylene imine monomers. PEIs are available in two forms - branched polyethylenimine (BPEI) and linear polyethylenimine (LPEI) with various molecular weights (from 200 to 800,000 Da). The similar to PAMAM, the transfection efficiency and cytotoxicity of PEI are dependent on their size. For example, high molecular PEI/ DNA formulations are more efficient and also much more toxic than low molecular PEI/DNA formulations.15 PAMAM and PEI with positive primary amine as surface groups is able to condense the negatively charged DNA into polyplexes (polymer/DNA complexes) with a net positive charge. These complexes bind to negatively charged cell membrane components by electrostatic interactions.16,17 After crossing the cellular membrane via the endocytotic pathway,18 Secondary and tertiary amines of PAMAM and PEI act as a “proton sponge”. The proton sponge effect induce the endosome disruption and polyplexes escape into the cytoplasm.19 As described above, PAMAM and PEI have the major problems such as cytotoxicity and relatively low transfection efficacy. Therefore, we conjugated nontoxic PEI layers with low molecular weight to a PAMAM core to achieve the enhanced gene transfection activity as well as to minimize the cytotoxicity. The physicochemical properties of PMPE/DNA complex were characterized. Biosafety and gene delivery efficiency of the PMPE/DNA complexes were also investigated in vitro.
Experimental Materials. Polyamidoamine dendrimer (PAMAM, generation 2,3,4), polyethylenimine (branched, 800 Da, 2 kDa, and 25 kDa), ethidium bromide, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), methyl acrylate, ethylenediamine, and methanol were purchased from Sigma-Aldrich (Seoul, South Korea). Luciferase 1000 assay kit and reporter lysis buffer were purchased from Promega (Madison, WI, USA). The luciferase expression plasmid DNA (pCN-Luci) was prepared as reported previously.20 293 and HeLa cell lines were obtained from Korean Cell Line Bank. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and 100× antibiotic-antimycotic reagent were purchased from GIBCO (Gaithersburg, MD, USA). Micro BCATM protein assay kit was purchased from Pierce (Rockford, IL, USA). Synthesis of Polyamidoamine-Methyl Acrylate-Polyethylenimine-Ethylenediamine (PMPE). PAMAM G2 and PAMAM G3 were dissolved in 1 mL of methanol with 3,200-fold and 6,400-fold molar ratios of methyl acrylate, respectively. The solution was stirred in a nitrogen atmosphere at 40 oC for 3 days, and the sample was evaporated under reduced pressure to remove the solvent and was quenched by the addition of an excess amount of cold ethyl ether. The precipitated sample (PAMAM G2-MA and PAMAM G3-MA) was dissolved in 2 mL of methanol with 1-fold molar ratios of PEI (800 Da, 758
2,000 Da), respectively. The solution was stirred in a nitrogen atmosphere at 40 oC for 2 days, and 1 mL of ethylenediamine was added, respectively. The solution was stirred in a nitrogen atmosphere at 40 oC for 1 day, and the sample was quenched by the addition of an excess amount of cold ethyl ether. The precipitated product was solubilized in water and dialyzed for 1 day against distilled water using dialysis membrane (MWCO 3,500, Spectra/Por® Spectrum Laboratories, CA, USA). The final product was freeze-dried and collected as a white solid powder. The 1H NMR spectra were measured using a Bruker DRX 300 (300 MHz) spectrometer and D2O was used as the solvent. Agarose Gel Retardation Assay. Complex formation of PMPE and plasmid DNA was examined by measuring the electrophoretic mobility of the complexes. PMPE/pDNA complexes was prepared at different weight ratios (polymer/pDNA) ranging from 0.5 to 6.0 in HEPES buffer (20 mM Hepes, 150 mM NaCl, pH 7.4). After 30 min incubation at room temperature for complex formation, the samples were subject to electrophoresis on a 0.7% (w/v) agarose gel with ethidium bromide (0.5 g/mL of the gel). Agarose gel electrophoresis typically required 30 min at 100 V and the gel was analyzed on a UV illuminator to show the location of the pDNA. Dynamic Light Scattering and -Potential Measurements. The size and surface charge (-potential) of PMPE 2-800, PMPE 3-800, PMPE 2-2000, and PMPE 3-2000/plasmid complex at 4:1 weight ratio were determined by the Zeta-potential & Particle size Analyzer ELS-Z (Photal, Otsuka Electronics, Japan) and the Zetasizer Nano ZS (Malvern Instruments, UK), respectively. For size measurements, average values were calculated with the data from thirty runs. For zeta-potential measurements, average values were calculated with the data from three runs. Cytotoxicity Assay. Evaluation of cytotoxicity was performed by the MTT assay in 293 and HeLa cells. Cells were seeded at a density of 2×104 cells/well in 96-well plate and were incubated for 1 day before adding the polymer. Cells were treated with PAMAM G3, PEI 25 kDa, PEI 800 Da, PEI 2 kDa, and the PMPE at various concentrations for 24 h at 37 oC. Then, 26 L of MTT stock solution (2 mg/mL) was added and incubated for 4 h at 37 oC. MTT-containing medium was removed and 150 L DMSO was added to each well to dissolve the formazan crystal formed by live cells. The absorbance of each well was read on a microplate reader (VersaMax, Molecular Devices, US) at 570 nm. All experiments were performed in quadruplicate. Cell Culture and Transfection Assay. Human embryonic kidney 293 cells and human epithelial carcinoma HeLa cells were cultured in DMEM with 10% FBS, 1% antibiotic-antimycotic agent. Cell lines were maintained in an incubator (5% CO2, 95% relative humidity, 37 oC). For the transfection study, cells were seeded in a 96-well plate at a density of 2×105 cells/well and incubated for 24 h to reach 70%-80% confluency. The transfection complex was prepared by mixing Macromol. Res., Vol. 22, No. 7, 2014
Polyethylenimine-Grafted Polyamidoamine Conjugates for Gene Delivery with High Efficiency and Low Cytotoxicity
0.5 g of plasmid DNA with various amounts of PMPE containing 10% FBS for 30 min at room temperature. To compare the transfection efficiency, PEI 25 kDa/pDNA (weight ratios (polymer/pDNA): 2:1-8:1, optimal condition) complexes were prepared as a control group. PAMAM G3/pDNA complexes, PEI 800 Da/pDNA complexes, PEI 2 kDa/pDNA complexes, and PMPE/pDNA complexes were prepared with weight ratios (polymer/pDNA) of 4:1 to 16:1 to optimize transfection condition. Then, cells were treated with the complex solution and incubated for 24 h at 37 oC. The old medium was removed and the cells were rinsed with DPBS (Dulbecco's phosphate buffered saline), and shaken for 30 min at room temperature in 100 L of Reporter lysis buffer (Promega, Madison, WI) was added to each well. The cells were harvested and transferred to microcentrifuge tubes. After 15 s of vortexing, the cells were centrifuged at 13,200 rpm for 10 min. Luciferase activity was measured using a Lumat LB 9507 instrument (Berthold Technology, Bad Wildbad, Germany), and protein content was measured using a Micro BCATM protein assay kit. The final values of luciferase were reported in terms of RLU/g total protein. All experiments were performed in triplicate. Statistical Analysis. The statistical analysis was performed using the unpaired Student’s t-test (GraphPad Prism 5). All results are given as the mean±standard deviation (SD). Differences between groups were considered statistically significant at p
