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Click modification of helical amylose by poly(l -lysine) dendrons for non-viral gene delivery
Materials Science and Engineering C 49 (2015) 485–492
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Click modification of helical amylose by poly(L-lysine) dendrons for non-viral gene delivery Jia-Dong Pang a,1, Bao-Xiong Zhuang b,1, Kaijin Mai a, Ru-Fu Chen b, Jie Wang b,⁎, Li-Ming Zhang a,⁎ a PCFM Lab and GDHPPC Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, China b Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102, China
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Article history: Received 6 October 2014 Received in revised form 13 November 2014 Accepted 4 January 2015 Available online 6 January 2015 Keywords: Amylose Poly(L-lysine) dendron Click conjugation Gene delivery Cytotoxicity
a b s t r a c t Although amylose as a naturally-occurring helical polysaccharide has been widely used for biomedical applications, few studies have dealt with its chemical modification for non-viral gene delivery. In this work, the click modification of amylose by poly(L-lysine) dendrons was carried out and then characterized by Fourier transform infrared spectroscopy, wide-angle X-ray diffraction and elemental analyses. Such a modified polysaccharide exhibited excellent ability to condense plasmid pMSCV-GFP-PARK2 into compact and spherical nanoparticles. Moreover, it displayed much lower cytotoxicity when compared to branched polyethylenimine (bPEI, 25 kDa), a commercially available gene vector. Similar to bPEI, it had a dose-dependent gene transfection activity in human embryonic kidney 293T cells, as observed by confocal laser scanning microscopy and flow cytometry. At each optimized N/P ratio, the percentage of transfected cells by this modified polysaccharide was found to be comparable to that by bPEI. Western blot and cell apoptosis analyses confirmed its effectiveness for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been growing interest in the chemical modification of naturally occurring polysaccharides for developing non-viral gene vectors [1–4]. For example, Thomas et al. [5] modified dextran by reacting with glycidyltrimethyl-ammonium chloride, and then complexed with pGL-3 plasmid DNA for the gene transfection in hepatocellular carcinoma cells (HepG2); Chang et al. [6] modified chitosan by conjugating with histidine, and then complexed with pCMV-Luc plasmid DNA for the gene transfection in human embryonic kidney cells (HEK293); Yang et al. [7] modified alpha-cyclodextrin by conjugating oligoethylenimine, and then complexed with pRL-CMV plasmid DNA for the gene transfection in mammalian cell lines; Thomsen et al. [8] modified pullulan by conjugating with spermine, and then complexed with plasmid DNA for the gene transfection in rat brain endothelial cells (RBE4s) and human brain microvascular endothelial cells (HBMECs). In contrast to non-viral gene vectors based on commonly used synthetic polymers such as polyethylenimine (PEI) [9] and ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (L.-M. Zhang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2015.01.011 0928-4931/© 2015 Elsevier B.V. All rights reserved.
poly((2-dimethyl amino)ethyl methacrylate) [10], these modified polysaccharide gene vectors provide specific advantages such as nontoxicity, good degradability and biocompatibility as well as their molecular diversity suitable for further modification or chemical conjugation. As a helical polysaccharide that is present in nature as a component of starch [11], amylose has been widely used for biomedical applications, including prodrug preparation [12], encapsulation of bovine hemoglobin [13] and oral drug delivery [14]. To our knowledge, however, there is no information available on the modification of amylose for gene delivery except for a recent publication [15], which described the synthesis of a cationic cycloamylose derivative by the enzymatic reaction of recombinant potato D-enzyme on amylose and subsequent conjugation with spermine for the interaction with plasmid DNA encoding firefly luciferase. In this work, we carried out for the first time the chemical modification of amylose by the click conjugation of poly(L-lysine) dendrons for the delivery of plasmid pMSCV-GFP-PARK2 to human embryonic kidney 293T cells. The resultant amylose derivative was studies in terms of its complexation with plasmid pMSCV-GFP-PARK2, cytotoxicity, as well as gene delivery and transfection in cultured cells. Meanwhile, branched polyethylenimine (bPEI, 25 kDa), which is one of the most potent synthetic gene delivery vectors because of its high transfection efficiency [16], was used for comparative studies in some cases in order
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Scheme 1. A route to the click modification of amylose by poly(L-lysine) dendons.
to investigate the feasibility of this amylose derivative as a non-viral gene vector. 2. Materials and methods 2.1. Materials Native amylose from potato (98%) was purchased from SigmaAldrich. 1-Azido-2,3-epoxypropane was synthesized by two-step reactions (please see the supplementary information: Synthesis and characterization of 1-azido-2,3-epoxypropane) in our lab according to the method reported previously by Pahimanolis et al. [17]. Propargyl focal point poly(L-lysine) dendron (PLLD-G3, generation = 3) was synthesized in our lab by divergent and convergent approaches, as reported
in our recent publication [18]. Copper sulfate (CuSO4·5H2O) and sodium ascorbate (99%) were purchased from Alfa Aesar. The Dulbecco's modified Eagle's medium (DMEM), trypsin–ethylenediaminetetraaceticacid (Trypsin–EDTA), fetal bovine serum (FBS) and deoxyribonuclease I (DNase I) were purchased from Gibco-BRL (Canada). Branched polyethylenimine (bPEI, 25 kDa), ethidium bromide (EB), and 3-[4, 5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma. Deoxyribonuclease I (DNaseI) was purchased from Fermentas Life Sciences. Plasmid pMSCV-GFP-PARK2, encoding a green fluorescent protein (GFP), was provided by Beijing Genomics Institution (China). Human embryonic kidney 293T cells were provided by Second Affiliated Hospital of Sun Yat-sen University. Other chemical reagents of analytical grade were obtained commercially and used directly.
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PLLD-G3 (0.29 mmol) and 0.05 g azidized amylose were added successively under magnetic stirring. After a clear solution was obtained, 18 mg CuSO4·5H2O and 65 mg sodium ascorbate were then added successively under nitrogen atmosphere. The resultant reaction mixture was heated to 40 °C for 48 h. After the reaction, the product was dialyzed in distilled water for 3 d (MWCO = 14000) and lyophilized to obtain the modified amylose with a yield of 65%. To confirm this click reaction, the infrared spectra were recorded on a PerkinElmer Paragon 1000 Infrared Spectrometer, and the X-ray diffraction (XRD) patterns were obtained by a Rigacu D/MAX 2200 VPC Diffractometer (Japan). Based on elemental analysis, the number of PLLD-G3 per 100 anhydroglucose units of amylose was determined to be 9.35. 2.4. Complexation of modified amylose with plasmid pMSCV-GFP-PARK2 and its characterization
Fig. 1. FTIR spectra of amylose, azidized amylose, PLLD-G3 and modified amylose.
2.2. Azidation of amylose by 1-azido-2, 3-epoxypropane The introduction of azide groups onto the backbone of amylose was carried out as follows: to 50 mL of 0.1 mol/L NaOH solution, 240 mg amylose (1.5 mmol) and 180 μL (1.5 mmol) of freshly prepared solution of 1-azido-2,3-epoxypropane were added successively under magnetic stirring. The obtained clear reaction mixture solution was stirred for 24 h at 30 °C in a closed vial. After the reaction, the product was dialyzed in distilled water for 3 d (MWCO = 14000) and lyophilized to obtain the azidized amylose with a yield of 82%. FTIR (PerkinElmer Paragon 1000 spectrometer) and 1H NMR (Bruker DPX-300 NMR spectrometer) analyses were used to confirm the formation of azidized amylose. FTIR (KBr, cm−1): 3460 cm−1 (νO\H, pyranose), 2106 cm−1 (azido groups), and 1022 cm−1 (νC\O, pyranose). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 5.60–4.50 (protons on anhydroglucose unit), 3.78 (\CH2N3), 3.61 (\CH2O\).
The complexation of modified amylose with plasmid pMSCV-GFPPARK2 in a phosphate buffer solution (PBS, pH 7.4) was carried out at various N/P ratios. The N/P value was determined as the ratio of moles of the amine groups of modified amylose to moles of phosphates of plasmid pMSCV-GFP-PARK2. For each complexation, the resultant suspension was homogenized by gentle vortexing for 10 s and then incubated at room temperature for 30 min before use. Agarose gel electrophoresis was used to investigate the complexation effectiveness under different conditions (N/P ratios, storage time, without and with DNase I). For each test, 10 μL of complex suspension was analyzed at 100 V on 1.0% agarose gel containing 0.5 mg/mL ethidium bromide, and pDNA bands were visualized by a UV lamp using a GelDoc system (NanoDrop 2000, Thermo). For the resultant modified amylose/plasmid pMSCV-GFP-PARK2 complexes, their particle size and zeta potentials were determined by a Zeta Potential Analyzer instrument (ZetaPALS,
2.3. Click reaction between azidized amylose and propargyl focal point poly(L-lysine) dendron The click reaction between the azidized amylose and PLLD-G3 was carried out as follows: to 10 mL dimethylsulfoxide (DMSO), 0.515 g
Fig. 2. The XRD patterns of amylose before and after the click modification.
Fig. 3. (a) Electrophoretic mobility of plasmid pMSCV-GFP-PARK2 (pDNA) in the modified amylose/pDNA complexes after the complexation for 30 min; (b) electrophoretic mobility of pDNA in the modified amylose/pDNA complexes after the complexation for 36 h; (c) electrophoretic mobility of pDNA alone, pDNA treated with DNase I (2 μg), and pDNA in the modified amylose/pDNA complexes treated with DNase I (2 μg). From left lane to right lane, pDNA amounts in the case of pDNA alone or pDNA treated with DNase I were the same to those in the polyplexes treated with DNase I.
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Fig. 4. (A) Particle size of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios; (B) zeta potentials of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios; (C) TEM images of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratio of 20; (D) TEM images of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratio of 40.
Brookhaven Instruments Corporation, USA). Prior to the measurements, the complexes were diluted to 1.0 mL by 0.15 M NaCl. The morphological examination of the complexes was performed using a JEM-2010HR high-resolution transmission electron microscope after counterstained with uranyl acetate. 2.5. Cell viability assay Human embryonic kidney 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C, 5% CO2, and 95% relative humidity. For cell viability assay, the cells were seeded in 96-well sterile flat-bottom plates at an initial density of 1000–10000 cells/well in 200 μl growth medium (appropriate number of cells needed for each well was determined from growth curves) and incubated for 3–5 d to ensure adherence. Upon surface attachment, the cells were treated with the modified amylose or bPEI solutions at varying concentrations and aqueous suspensions of modified amylose/plasmid pMSCV-GFP-PARK2 or bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios. After the incubation for 4 h, 20 μl of MTT solution with the concentration of 5 mg/ml was added to each well. The reaction product was solubilized with 150 μl of DMSO under stirring for 10 min. The absorbance (A490) was measured by a Benchmark Plus Microplate Spectrophotometer at 490 nm. The cell viability (%) was calculated according to the following equation: cell viability (%) = [A490 (sample) / A490 (control)] × 100, where A490 (sample) was obtained in the presence of samples and A490 (control) was obtained in the absence of samples. 2.6. In vitro transfection assay In vitro transfection assay was carried out in 293T cells using plasmid pMSCV-GFP-PARK2 as reporter gene. In brief, 24-well plates were seeded with cells at a density of 1 × 105/well. After incubation for 12 h
(to reach 80% confluence at the time of transfection), the media were replaced with serum-free or 10% serum containing media with the modified amylose/plasmid pMSCV-GFP-PARK2 or bPEI/plasmid pMSCV-GFPPARK2 complexes at various N/P ratios and additionally incubated for 6 h. Then the media were exchanged for fresh media containing 10% serum and allowed to incubate for 24 h at 37 °C. After the incubation, the cells were observed with an Olympus IX71 fluorescence microscope (Melville, NY, U.S.A.). The transfected cells were washed once with PBS and detached with 0.25% trypsin. Transfection efficiency was evaluated by scoring the percentage of cells expressing GFP, using a FACS Aria flow cytometer (Germany).
2.7. Western blot analysis Equal amounts of extracted protein as determined from Bradfords' assay were separated on 12.5% polyacrylamide gel and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were incubated with appropriate anti-rabbit conjugated IgG (Sigma-Aldrich) at a dilution of 1:3000 for 1 h at room temperature. Detection was performed using a Super Signal West Femto chemiluminescence reagent (Pierce, Thermo Scientific) as per the manufacturer's instructions.
2.8. Cellular apoptosis assay The cells after the treatments under various conditions were quickly trypsinized, detached from plastic plate, and washed with PBS for two times. The cells were then suspended with HEPES buffer and stained with Annexin V and propidium iodide for 15 min as directed by the manufacturer's protocol (Lianke, China). The stained cells were assessed with an FACS Aria flow cytometer.
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crystallinity peak was almost not present. Moreover, the diffraction intensity was greatly weakened after the click reaction. These results could be attributed to the disruption of amylose crystalline structure by the click conjugation of poly(L-lysine) dendron, which confirmed further the success of the click reaction between PLLD-G3 dendron and azidized amylose. 3.2. Formation of modified amylose/plasmid pMSCV-GFP-PARK2 complexes and their physicochemical characteristics
Fig. 5. The in vitro cytotoxicity of modified amylose and bPEI at various concentrations (A) and their complexes with plasmid pMSCV-GFP-PARK2 (pDNA) at various N/P ratios (B). Cell viability was determined by the MTT assay and expressed as a percentage of control (untreated cell cultures).
3. Results and discussion 3.1. Click modification of amylose by poly(L-lysine) dendrons For the click modification of amylose, amylose was firstly functionalized by 1-azido-2,3-epoxypropane to introduce the azide groups, and then conjugated with propargyl focal point poly(L-lysine) dendron of third generation (PLLD-G3) by a copper-catalyzed azide alkyne cyclization reaction, as shown in Scheme 1. Fig. 1 gives the FTIR spectra of amylose, azidized amylose, PLLD-G3 and modified amylose. In comparison with the spectrum of amylose, the spectrum of azidized amylose showed a new strong peak at 2106 cm−1, confirming the introduction of azide group onto amylose backbone. The characteristic absorption bands of PLLD-G3 appeared at 2917 cm− 1, 2842 cm− 1 (νC\H), 1604 cm− 1 (νC_O) and 1396 cm− 1 (νCO\NH). The characteristic absorption bands of azidized amylose appeared at 3460 cm−1 (νO\H, pyranose), 2106 cm− 1 (azido groups), and 1022 cm−1 (νC\O, pyranose). After the click modification, the spectrum of the modified amylose did not show the characteristic absorption bands of azido group (2106 cm−1), but exhibited the main characteristic bands of PLLD-G3 and azidized amylose. Another evidence for the click reaction was provided by XRD analyses. Fig. 2 shows the XRD patterns of amylose before and after the click reaction. Before the click reaction, the XRD pattern displayed a strong reflection at 2θ about 17° due to high crystallization in the molecular chains of amylose. After the click reaction, however, this strong
One prerequisite of the modified amylose as a non-viral gene vector is its ability to condense plasmid pMSCV-GFP-PARK2 into a particulate structure. To investigate this, we carried out agarose gel electrophoresis, particle size and zeta potential measurements, as well as TEM observation for aqueous modified amylose/plasmid pMSCV-GFP-PARK2 mixed systems. Fig. 3a gives the electrophoretic mobility of plasmid pMSCV-GFPPARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes prepared after the complexation for 30 min at various N/P ratios (0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, and 60). As seen, the migration of plasmid pMSCV-GFP-PARK2 was completely retarded when the N/P ratio of modified amylose/plasmid pMSCV-GFP-PARK2 complexes was equal to or greater than 2, which confirmed the binding ability of modified amylose to plasmid pMSCV-GFP-PARK2 and the formation of modified amylose/plasmid pMSCV-GFP-PARK2 complexes. Moreover, the binding ability of modified amylose to plasmid pMSCV-GFP-PARK2 could be kept after the complexation for 36 h, as shown in Fig. 3b. In addition, the effect of DNase I on the complexation of modified amylose with plasmid pMSCV-GFP-PARK2 was also investigated by agarose gel electrophoresis. Fig. 3c gives the electrophoretic mobility of plasmid pMSCV-GFP-PARK2 alone, plasmid pMSCV-GFP-PARK2 treated with DNase I, and plasmid pMSCV-GFP-PARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes treated with DNase I. In contrast to naked plasmid pMSCV-GFP-PARK2 as the control, plasmid pMSCV-GFPPARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes was protected from degradation by DNase I. This implies that more integral plasmid pMSCV-GFP-PARK2 may transfer to cells without degradation by enzymes when the modified amylose is used for gene delivery. Fig. 4 shows the particle size and zeta potentials of modified amylose/plasmid pMSCV-GFP-PARK2 complexes formed when the N/P ratio is equal to or greater than 2 as well as TEM images of modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratios of 20 and 40. From Fig. 4A, the mean particle size was observed to have an obvious decrease from 731 to 282 nm with the increase of the N/P ratio from 2 to 10, and then remained in the size range from 200 to 240 nm with
Fig. 6. In vitro gene transfection efficiencies of the modified amylose/plasmid pMSCV-GFPPARK2 complexes in comparison with those of bPEI/plasmid pMSCV-GFP-PARK2 complexes in the presence of 10% serum. Data represent mean ± standard deviation (n = 3).
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Fig. 7. Typical fluorescence images of 293T cells transfected by the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios.
further increase of the N/P ratio. This is because the modified amylose complexes with plasmid pMSCV-GFP-PARK2 through ionic interactions. At a low N/P ratio of 2, the complexes could not form completely and therefore had a large hydrodynamic size. At high N/P ratios, there are net electrostatic repulsive forces to prevent aggregation among the complexes. From Fig. 4B, the zeta potential was found to have an obvious increase from − 5.9 to + 10.8 with the increase of N/P ratio from 2 to 10, and then remained to be about +13.0 with further increase of N/P ratio. In this case, the highly negative charge of plasmid pMSCVGFP-PARK2 is neutralized rapidly when the modified amylose is added, and the surface charge of the complexes becomes positive at higher N/P ratios. It is known [19,20] that a positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates cellular uptake. In addition, the morphology of
modified amylose/plasmid pMSCV-GFP-PARK2 complexes was investigated by TEM observation at two N/P ratios and found to have a spherical shape and compacted structure, as shown in Fig. 4C and D. 3.3. Cytotoxicity of modified amylose and its complexes with plasmid pMSCV-GFP-PARK2 The in vitro cytotoxicity of modified amylose in the concentration range from 14 to 275 μg/mL and its complexes with plasmid pMSCVGFP-PARK2 at various N/P ratios were evaluated in 293T cells by MTT assays in comparison with that of commercially available bPEI, as shown in Fig. 5. For bPEI (25 kDa) and its complexes with plasmid pMSCV-GFP-PARK2, significant cytotoxicity was observed when bPEI concentration was higher than 27.5 μg/mL and the N/P ratio was higher
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which will become an advantage when such a modified polysaccharide derivative is used for gene delivery. 3.4. Transfection effectiveness of modified amylose for plasmid pMSCV-GFPPARK2 delivery
Fig. 8. Western blot analyses of PARK2 protein expressions in 293T cells treated with plasmid pMSCV-GFP-PARK2 only (A), the modified amylose/plasmid pMSCV-GFP- PARK2 complexes (N/P = 30, B) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10, C), respectively. GAPDH was used as an internal standard for the protein loading.
than 5. In these cases, the cell viability was found to be lower than 30%. In contrast, the modified amylose and its complexes with plasmid pMSCV-GFP-PARK2 could exhibit high cell viability, even at high concentrations and N/P ratios. At the concentration of 110 μg/mL and the N/P ratio of 40, for example, the cell viability was found to be about 90% for the modified amylose and its complexes with plasmid pMSCVGFP-PARK2. Obviously, the modified amylose displayed much lower cytotoxicity when compared to bPEI, a commonly used gene vector. These results are attributed to good biocompatibity of the modified amylose,
The in vitro transfection efficiencies of modified amylose or bPEI for plasmid pMSCV-GFP-PARK2 delivery were assessed in 293T cells, as shown in Fig. 6. Depending on the N/P ratio, the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFPPARK2 complexes have various transfection efficiencies. For the modified amylose/plasmid pMSCV-GFP-PARK2 complexes, the transfection efficiency increased with the increase of N/P ratio from 10 to 30, and had a slight decrease with a further increase of N/P ratio to 40. In contrast, the transfection efficiency of bPEI/plasmid pMSCV-GFP-PARK2 complexes decreased continuously with the increase of N/P ratio from 10 to 40. A decrease of transfection efficiency at a higher N/P ratio could be attributed to the cytotoxicity [21]. In particular, an optimized N/P ratio corresponding to the maximum transfection efficiency was found to be 30 for the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and 10 for bPEI/ plasmid pMSCV-GFP-PARK2 complexes, respectively. Fig. 7 gives typical fluorescence images of 293T cells transfected by the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios. As seen, the effects of N/P ratio on the fluorescence signal intensity in each case were consistent with the effects of N/P ratio on the transfection efficiency (Fig. 6). Similarly, the strongest fluorescence signal was observed at the N/P ratio of 30 for the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and 10 for bPEI/plasmid pMSCV-GFPPARK2 complexes, respectively.
Fig. 9. Cell apoptosis analyses by flow cytometry for 293T cells treated with treated with plasmid pMSCV-GFP-PARK2 only (A), the modified amylose/plasmid pMSCV-GFP- PARK2 complexes (N/P = 30, B) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10, C), respectively.
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To confirm the effectiveness of modified amylose for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells, Western blot and cell apoptosis analyses were also carried out. Fig. 8 shows Western blot analyses of PARK2 protein expressions in 293T cells incubated with plasmid pMSCV-GFP-PARK2 only, the modified amylose/plasmid pMSCV-GFPPARK2 complexes (N/P = 30) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10), respectively. In the case of plasmid pMSCVGFP-PARK2 only, few PARK2 protein expressions were observed. In contrast, a high level of PARK2 protein expressions were found in the case of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes (N/ P = 30) or bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10). It is known [22] that the increase in the expression of a particular protein reported is an indirect proof of successful transfection of a specific gene. Therefore, the modified amylose has good gene delivery capability similar to commonly used bPEI gene vector. Moreover, the use of this modified amylose did not induce significant cellular apoptosis when compared to bPEI, as illustrated in Fig. 9. 4. Conclusions Although amylose has been widely used for biomedical applications, few studies have dealt with its modification for efficient gene delivery. In this work, we modified amylose by the click conjugation with propargyl focal point poly(L-lysine) dendron of third generation and then used successfully for the delivery of plasmid pMSCV-GFP-PARK2 to human embryonic kidney 293T cells. This novel amylose derivative containing poly(L-lysine) dendrons is characteristic of much lower cytotoxicity and considerable gene delivery capability when compared to branched polyethylenimine (bPEI, 25 kDa), a commonly used gene vector. Considering the supramolecular encapsulation of helical amylose to singlewall carbon nanotubes [23] or drugs [24], our next work will focus on the potential applications of such a polysaccharide conjugate for combined gene and photothermal therapy as well as gene and drug codelivery in order to enhance the therapeutic efficacy. Acknowledgments This work was supported by National Natural Science Foundation of China (51273216, 21074152, J1103305), Doctoral Research Program of Education Ministry in China (20090171110023 and 20130171120095),
Key Project of Scientific and Technical Innovation for Universities in Guangdong Province (cxzd1102) and Natural Science Foundation of Guangdong Province (S2013010012549 and S2013040014401). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.01.011. References [1] K. Raemdonck, T.F. Martens, K. Braeckmans, J. Demeester, S.C. De Smedt, Adv. Drug Deliv. Rev. 65 (2013) 1123. [2] M.D. Buschmann, A. Merzouki, M. Lavertu, M. Thibault, M. Jean, V. Darras, Adv. Drug Deliv. Rev. 65 (2013) 1234. [3] W. Khan, H. Hosseinkhani, D. Ickowicz, P.D. Hong, D.S. Yu, A.J. Domb, Acta Biomater. 8 (2012) 4224. [4] L. Casettari, D. Vllasaliu, J.K. Lam, M. Soliman, L. Illum, Biomaterials 33 (2012) 7565. [5] J.J. Thomas, M.R. Rekha, C.P. Sharma, Int. J. Pharm. 389 (2010) 195. [6] K.L. Chang, Y. Higuchi, S. Kawakami, F. Yamashita, M. Hashida, Bioconjug. Chem. 21 (2010) 1087. [7] C. Yang, H. Li, S.H. Goh, J. Li, Biomaterials 28 (2007) 3245. [8] L.B. Thomsen, J. Lichota, K.S. Kim, T. Moos, J. Control. Release 151 (2011) 45. [9] W.T. Godbey, K.K. Wu, A.G. Mikos, J. Control. Release 60 (1999) 149. [10] F.J. Verbaan, C. Oussoren, I.M. van Dam, Y. Takakura, M. Hashida, D.J. Crommelin, W.E. Hennink, G. Storm, Int. J. Pharm. 214 (2001) 99 (G.). [11] H.C. Wu, A. Sarko, Carbohydr. Res. 61 (1978) 27. [12] X. Cai, L. Yang, L.M. Zhang, Q. Wu, Carbohydr. Res. 345 (2010) 922. [13] W. Gao, B. Sha, W. Zou, X. Liang, X. Meng, H. Xu, J. Tang, D. Wu, L. Xu, H. Zhang, Biomaterials 32 (2011) 9425. [14] S.H. Moghadam, H.W. Wang, E. Saddar El-Leithy, C. Chebli, L. Cartilier, Biomed. Mater. 2 (2007) S71. [15] S. Toita, N. Morimoto, K. Akiyoshi, Biomacromolecules 11 (2010) 397. [16] S. Patnaik, K.C. Gupta, Expert Opin. Drug Deliv. 10 (2013) 215. [17] N. Pahimanolis, A.H. Vesterinen, J. Rich, J. Seppala, Carbohydr. Polym. 82 (2010) 78. [18] D. Ma, H.B. Zhang, Y.Y. Chen, J.T. Lin, L.M. Zhang, J. Colloid Interface Sci. 405 (2013) 305. [19] K. Kunath, A. von Harpe, D. Fischer, H. Petersen, U. Bickel, K. Voigt, T. Kissel, J. Control. Release 89 (2003) 113 (T). [20] H. Petersen, P.M. Fechner, A.L. Martin, K. Kunath, S. Stolnik, C.L. Roberts, D. Fischer, M.C. Davies, T. Kissel, Bioconjug. Chem. 13 (2002) 845. [21] H.L. Jiang, Y.K. Kim, R. Arote, J.W. Nah, M.H. Cho, W.J. Choi, T. Akaike, C.S. Cho, J. Control. Release 117 (2007) 273. [22] A. Bajaj, P. Kondaiah, S. Bhattacharya, J. Med. Chem. 50 (2007) 2432. [23] O.K. Kim, J. Je, J.W. Baldwin, S. Kooi, P.E. Pehrsson, L.J. Buckley, J. Am. Chem. Soc. 125 (2003) 4426. [24] B.G. Li, L.M. Zhang, Prog. Chem. 22 (2010) 1161.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Click modification of helical amylose by poly(L-lysine) dendrons for non-viral gene delivery Jia-Dong Pang a,1, Bao-Xiong Zhuang b,1, Kaijin Mai a, Ru-Fu Chen b, Jie Wang b,⁎, Li-Ming Zhang a,⁎ a PCFM Lab and GDHPPC Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, China b Second Affiliated Hospital, Sun Yat-sen (Zhongshan) University, Guangzhou 510102, China
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Article history: Received 6 October 2014 Received in revised form 13 November 2014 Accepted 4 January 2015 Available online 6 January 2015 Keywords: Amylose Poly(L-lysine) dendron Click conjugation Gene delivery Cytotoxicity
a b s t r a c t Although amylose as a naturally-occurring helical polysaccharide has been widely used for biomedical applications, few studies have dealt with its chemical modification for non-viral gene delivery. In this work, the click modification of amylose by poly(L-lysine) dendrons was carried out and then characterized by Fourier transform infrared spectroscopy, wide-angle X-ray diffraction and elemental analyses. Such a modified polysaccharide exhibited excellent ability to condense plasmid pMSCV-GFP-PARK2 into compact and spherical nanoparticles. Moreover, it displayed much lower cytotoxicity when compared to branched polyethylenimine (bPEI, 25 kDa), a commercially available gene vector. Similar to bPEI, it had a dose-dependent gene transfection activity in human embryonic kidney 293T cells, as observed by confocal laser scanning microscopy and flow cytometry. At each optimized N/P ratio, the percentage of transfected cells by this modified polysaccharide was found to be comparable to that by bPEI. Western blot and cell apoptosis analyses confirmed its effectiveness for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been growing interest in the chemical modification of naturally occurring polysaccharides for developing non-viral gene vectors [1–4]. For example, Thomas et al. [5] modified dextran by reacting with glycidyltrimethyl-ammonium chloride, and then complexed with pGL-3 plasmid DNA for the gene transfection in hepatocellular carcinoma cells (HepG2); Chang et al. [6] modified chitosan by conjugating with histidine, and then complexed with pCMV-Luc plasmid DNA for the gene transfection in human embryonic kidney cells (HEK293); Yang et al. [7] modified alpha-cyclodextrin by conjugating oligoethylenimine, and then complexed with pRL-CMV plasmid DNA for the gene transfection in mammalian cell lines; Thomsen et al. [8] modified pullulan by conjugating with spermine, and then complexed with plasmid DNA for the gene transfection in rat brain endothelial cells (RBE4s) and human brain microvascular endothelial cells (HBMECs). In contrast to non-viral gene vectors based on commonly used synthetic polymers such as polyethylenimine (PEI) [9] and ⁎ Corresponding authors. E-mail addresses: [email protected] (J. Wang), [email protected] (L.-M. Zhang). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.msec.2015.01.011 0928-4931/© 2015 Elsevier B.V. All rights reserved.
poly((2-dimethyl amino)ethyl methacrylate) [10], these modified polysaccharide gene vectors provide specific advantages such as nontoxicity, good degradability and biocompatibility as well as their molecular diversity suitable for further modification or chemical conjugation. As a helical polysaccharide that is present in nature as a component of starch [11], amylose has been widely used for biomedical applications, including prodrug preparation [12], encapsulation of bovine hemoglobin [13] and oral drug delivery [14]. To our knowledge, however, there is no information available on the modification of amylose for gene delivery except for a recent publication [15], which described the synthesis of a cationic cycloamylose derivative by the enzymatic reaction of recombinant potato D-enzyme on amylose and subsequent conjugation with spermine for the interaction with plasmid DNA encoding firefly luciferase. In this work, we carried out for the first time the chemical modification of amylose by the click conjugation of poly(L-lysine) dendrons for the delivery of plasmid pMSCV-GFP-PARK2 to human embryonic kidney 293T cells. The resultant amylose derivative was studies in terms of its complexation with plasmid pMSCV-GFP-PARK2, cytotoxicity, as well as gene delivery and transfection in cultured cells. Meanwhile, branched polyethylenimine (bPEI, 25 kDa), which is one of the most potent synthetic gene delivery vectors because of its high transfection efficiency [16], was used for comparative studies in some cases in order
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Scheme 1. A route to the click modification of amylose by poly(L-lysine) dendons.
to investigate the feasibility of this amylose derivative as a non-viral gene vector. 2. Materials and methods 2.1. Materials Native amylose from potato (98%) was purchased from SigmaAldrich. 1-Azido-2,3-epoxypropane was synthesized by two-step reactions (please see the supplementary information: Synthesis and characterization of 1-azido-2,3-epoxypropane) in our lab according to the method reported previously by Pahimanolis et al. [17]. Propargyl focal point poly(L-lysine) dendron (PLLD-G3, generation = 3) was synthesized in our lab by divergent and convergent approaches, as reported
in our recent publication [18]. Copper sulfate (CuSO4·5H2O) and sodium ascorbate (99%) were purchased from Alfa Aesar. The Dulbecco's modified Eagle's medium (DMEM), trypsin–ethylenediaminetetraaceticacid (Trypsin–EDTA), fetal bovine serum (FBS) and deoxyribonuclease I (DNase I) were purchased from Gibco-BRL (Canada). Branched polyethylenimine (bPEI, 25 kDa), ethidium bromide (EB), and 3-[4, 5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma. Deoxyribonuclease I (DNaseI) was purchased from Fermentas Life Sciences. Plasmid pMSCV-GFP-PARK2, encoding a green fluorescent protein (GFP), was provided by Beijing Genomics Institution (China). Human embryonic kidney 293T cells were provided by Second Affiliated Hospital of Sun Yat-sen University. Other chemical reagents of analytical grade were obtained commercially and used directly.
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PLLD-G3 (0.29 mmol) and 0.05 g azidized amylose were added successively under magnetic stirring. After a clear solution was obtained, 18 mg CuSO4·5H2O and 65 mg sodium ascorbate were then added successively under nitrogen atmosphere. The resultant reaction mixture was heated to 40 °C for 48 h. After the reaction, the product was dialyzed in distilled water for 3 d (MWCO = 14000) and lyophilized to obtain the modified amylose with a yield of 65%. To confirm this click reaction, the infrared spectra were recorded on a PerkinElmer Paragon 1000 Infrared Spectrometer, and the X-ray diffraction (XRD) patterns were obtained by a Rigacu D/MAX 2200 VPC Diffractometer (Japan). Based on elemental analysis, the number of PLLD-G3 per 100 anhydroglucose units of amylose was determined to be 9.35. 2.4. Complexation of modified amylose with plasmid pMSCV-GFP-PARK2 and its characterization
Fig. 1. FTIR spectra of amylose, azidized amylose, PLLD-G3 and modified amylose.
2.2. Azidation of amylose by 1-azido-2, 3-epoxypropane The introduction of azide groups onto the backbone of amylose was carried out as follows: to 50 mL of 0.1 mol/L NaOH solution, 240 mg amylose (1.5 mmol) and 180 μL (1.5 mmol) of freshly prepared solution of 1-azido-2,3-epoxypropane were added successively under magnetic stirring. The obtained clear reaction mixture solution was stirred for 24 h at 30 °C in a closed vial. After the reaction, the product was dialyzed in distilled water for 3 d (MWCO = 14000) and lyophilized to obtain the azidized amylose with a yield of 82%. FTIR (PerkinElmer Paragon 1000 spectrometer) and 1H NMR (Bruker DPX-300 NMR spectrometer) analyses were used to confirm the formation of azidized amylose. FTIR (KBr, cm−1): 3460 cm−1 (νO\H, pyranose), 2106 cm−1 (azido groups), and 1022 cm−1 (νC\O, pyranose). 1H NMR (300 MHz, DMSO-d6): δ (ppm) = 5.60–4.50 (protons on anhydroglucose unit), 3.78 (\CH2N3), 3.61 (\CH2O\).
The complexation of modified amylose with plasmid pMSCV-GFPPARK2 in a phosphate buffer solution (PBS, pH 7.4) was carried out at various N/P ratios. The N/P value was determined as the ratio of moles of the amine groups of modified amylose to moles of phosphates of plasmid pMSCV-GFP-PARK2. For each complexation, the resultant suspension was homogenized by gentle vortexing for 10 s and then incubated at room temperature for 30 min before use. Agarose gel electrophoresis was used to investigate the complexation effectiveness under different conditions (N/P ratios, storage time, without and with DNase I). For each test, 10 μL of complex suspension was analyzed at 100 V on 1.0% agarose gel containing 0.5 mg/mL ethidium bromide, and pDNA bands were visualized by a UV lamp using a GelDoc system (NanoDrop 2000, Thermo). For the resultant modified amylose/plasmid pMSCV-GFP-PARK2 complexes, their particle size and zeta potentials were determined by a Zeta Potential Analyzer instrument (ZetaPALS,
2.3. Click reaction between azidized amylose and propargyl focal point poly(L-lysine) dendron The click reaction between the azidized amylose and PLLD-G3 was carried out as follows: to 10 mL dimethylsulfoxide (DMSO), 0.515 g
Fig. 2. The XRD patterns of amylose before and after the click modification.
Fig. 3. (a) Electrophoretic mobility of plasmid pMSCV-GFP-PARK2 (pDNA) in the modified amylose/pDNA complexes after the complexation for 30 min; (b) electrophoretic mobility of pDNA in the modified amylose/pDNA complexes after the complexation for 36 h; (c) electrophoretic mobility of pDNA alone, pDNA treated with DNase I (2 μg), and pDNA in the modified amylose/pDNA complexes treated with DNase I (2 μg). From left lane to right lane, pDNA amounts in the case of pDNA alone or pDNA treated with DNase I were the same to those in the polyplexes treated with DNase I.
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Fig. 4. (A) Particle size of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios; (B) zeta potentials of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios; (C) TEM images of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratio of 20; (D) TEM images of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratio of 40.
Brookhaven Instruments Corporation, USA). Prior to the measurements, the complexes were diluted to 1.0 mL by 0.15 M NaCl. The morphological examination of the complexes was performed using a JEM-2010HR high-resolution transmission electron microscope after counterstained with uranyl acetate. 2.5. Cell viability assay Human embryonic kidney 293T cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C, 5% CO2, and 95% relative humidity. For cell viability assay, the cells were seeded in 96-well sterile flat-bottom plates at an initial density of 1000–10000 cells/well in 200 μl growth medium (appropriate number of cells needed for each well was determined from growth curves) and incubated for 3–5 d to ensure adherence. Upon surface attachment, the cells were treated with the modified amylose or bPEI solutions at varying concentrations and aqueous suspensions of modified amylose/plasmid pMSCV-GFP-PARK2 or bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios. After the incubation for 4 h, 20 μl of MTT solution with the concentration of 5 mg/ml was added to each well. The reaction product was solubilized with 150 μl of DMSO under stirring for 10 min. The absorbance (A490) was measured by a Benchmark Plus Microplate Spectrophotometer at 490 nm. The cell viability (%) was calculated according to the following equation: cell viability (%) = [A490 (sample) / A490 (control)] × 100, where A490 (sample) was obtained in the presence of samples and A490 (control) was obtained in the absence of samples. 2.6. In vitro transfection assay In vitro transfection assay was carried out in 293T cells using plasmid pMSCV-GFP-PARK2 as reporter gene. In brief, 24-well plates were seeded with cells at a density of 1 × 105/well. After incubation for 12 h
(to reach 80% confluence at the time of transfection), the media were replaced with serum-free or 10% serum containing media with the modified amylose/plasmid pMSCV-GFP-PARK2 or bPEI/plasmid pMSCV-GFPPARK2 complexes at various N/P ratios and additionally incubated for 6 h. Then the media were exchanged for fresh media containing 10% serum and allowed to incubate for 24 h at 37 °C. After the incubation, the cells were observed with an Olympus IX71 fluorescence microscope (Melville, NY, U.S.A.). The transfected cells were washed once with PBS and detached with 0.25% trypsin. Transfection efficiency was evaluated by scoring the percentage of cells expressing GFP, using a FACS Aria flow cytometer (Germany).
2.7. Western blot analysis Equal amounts of extracted protein as determined from Bradfords' assay were separated on 12.5% polyacrylamide gel and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were incubated with appropriate anti-rabbit conjugated IgG (Sigma-Aldrich) at a dilution of 1:3000 for 1 h at room temperature. Detection was performed using a Super Signal West Femto chemiluminescence reagent (Pierce, Thermo Scientific) as per the manufacturer's instructions.
2.8. Cellular apoptosis assay The cells after the treatments under various conditions were quickly trypsinized, detached from plastic plate, and washed with PBS for two times. The cells were then suspended with HEPES buffer and stained with Annexin V and propidium iodide for 15 min as directed by the manufacturer's protocol (Lianke, China). The stained cells were assessed with an FACS Aria flow cytometer.
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crystallinity peak was almost not present. Moreover, the diffraction intensity was greatly weakened after the click reaction. These results could be attributed to the disruption of amylose crystalline structure by the click conjugation of poly(L-lysine) dendron, which confirmed further the success of the click reaction between PLLD-G3 dendron and azidized amylose. 3.2. Formation of modified amylose/plasmid pMSCV-GFP-PARK2 complexes and their physicochemical characteristics
Fig. 5. The in vitro cytotoxicity of modified amylose and bPEI at various concentrations (A) and their complexes with plasmid pMSCV-GFP-PARK2 (pDNA) at various N/P ratios (B). Cell viability was determined by the MTT assay and expressed as a percentage of control (untreated cell cultures).
3. Results and discussion 3.1. Click modification of amylose by poly(L-lysine) dendrons For the click modification of amylose, amylose was firstly functionalized by 1-azido-2,3-epoxypropane to introduce the azide groups, and then conjugated with propargyl focal point poly(L-lysine) dendron of third generation (PLLD-G3) by a copper-catalyzed azide alkyne cyclization reaction, as shown in Scheme 1. Fig. 1 gives the FTIR spectra of amylose, azidized amylose, PLLD-G3 and modified amylose. In comparison with the spectrum of amylose, the spectrum of azidized amylose showed a new strong peak at 2106 cm−1, confirming the introduction of azide group onto amylose backbone. The characteristic absorption bands of PLLD-G3 appeared at 2917 cm− 1, 2842 cm− 1 (νC\H), 1604 cm− 1 (νC_O) and 1396 cm− 1 (νCO\NH). The characteristic absorption bands of azidized amylose appeared at 3460 cm−1 (νO\H, pyranose), 2106 cm− 1 (azido groups), and 1022 cm−1 (νC\O, pyranose). After the click modification, the spectrum of the modified amylose did not show the characteristic absorption bands of azido group (2106 cm−1), but exhibited the main characteristic bands of PLLD-G3 and azidized amylose. Another evidence for the click reaction was provided by XRD analyses. Fig. 2 shows the XRD patterns of amylose before and after the click reaction. Before the click reaction, the XRD pattern displayed a strong reflection at 2θ about 17° due to high crystallization in the molecular chains of amylose. After the click reaction, however, this strong
One prerequisite of the modified amylose as a non-viral gene vector is its ability to condense plasmid pMSCV-GFP-PARK2 into a particulate structure. To investigate this, we carried out agarose gel electrophoresis, particle size and zeta potential measurements, as well as TEM observation for aqueous modified amylose/plasmid pMSCV-GFP-PARK2 mixed systems. Fig. 3a gives the electrophoretic mobility of plasmid pMSCV-GFPPARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes prepared after the complexation for 30 min at various N/P ratios (0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, and 60). As seen, the migration of plasmid pMSCV-GFP-PARK2 was completely retarded when the N/P ratio of modified amylose/plasmid pMSCV-GFP-PARK2 complexes was equal to or greater than 2, which confirmed the binding ability of modified amylose to plasmid pMSCV-GFP-PARK2 and the formation of modified amylose/plasmid pMSCV-GFP-PARK2 complexes. Moreover, the binding ability of modified amylose to plasmid pMSCV-GFP-PARK2 could be kept after the complexation for 36 h, as shown in Fig. 3b. In addition, the effect of DNase I on the complexation of modified amylose with plasmid pMSCV-GFP-PARK2 was also investigated by agarose gel electrophoresis. Fig. 3c gives the electrophoretic mobility of plasmid pMSCV-GFP-PARK2 alone, plasmid pMSCV-GFP-PARK2 treated with DNase I, and plasmid pMSCV-GFP-PARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes treated with DNase I. In contrast to naked plasmid pMSCV-GFP-PARK2 as the control, plasmid pMSCV-GFPPARK2 in the modified amylose/plasmid pMSCV-GFP-PARK2 complexes was protected from degradation by DNase I. This implies that more integral plasmid pMSCV-GFP-PARK2 may transfer to cells without degradation by enzymes when the modified amylose is used for gene delivery. Fig. 4 shows the particle size and zeta potentials of modified amylose/plasmid pMSCV-GFP-PARK2 complexes formed when the N/P ratio is equal to or greater than 2 as well as TEM images of modified amylose/plasmid pMSCV-GFP-PARK2 complexes at the N/P ratios of 20 and 40. From Fig. 4A, the mean particle size was observed to have an obvious decrease from 731 to 282 nm with the increase of the N/P ratio from 2 to 10, and then remained in the size range from 200 to 240 nm with
Fig. 6. In vitro gene transfection efficiencies of the modified amylose/plasmid pMSCV-GFPPARK2 complexes in comparison with those of bPEI/plasmid pMSCV-GFP-PARK2 complexes in the presence of 10% serum. Data represent mean ± standard deviation (n = 3).
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Fig. 7. Typical fluorescence images of 293T cells transfected by the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios.
further increase of the N/P ratio. This is because the modified amylose complexes with plasmid pMSCV-GFP-PARK2 through ionic interactions. At a low N/P ratio of 2, the complexes could not form completely and therefore had a large hydrodynamic size. At high N/P ratios, there are net electrostatic repulsive forces to prevent aggregation among the complexes. From Fig. 4B, the zeta potential was found to have an obvious increase from − 5.9 to + 10.8 with the increase of N/P ratio from 2 to 10, and then remained to be about +13.0 with further increase of N/P ratio. In this case, the highly negative charge of plasmid pMSCVGFP-PARK2 is neutralized rapidly when the modified amylose is added, and the surface charge of the complexes becomes positive at higher N/P ratios. It is known [19,20] that a positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates cellular uptake. In addition, the morphology of
modified amylose/plasmid pMSCV-GFP-PARK2 complexes was investigated by TEM observation at two N/P ratios and found to have a spherical shape and compacted structure, as shown in Fig. 4C and D. 3.3. Cytotoxicity of modified amylose and its complexes with plasmid pMSCV-GFP-PARK2 The in vitro cytotoxicity of modified amylose in the concentration range from 14 to 275 μg/mL and its complexes with plasmid pMSCVGFP-PARK2 at various N/P ratios were evaluated in 293T cells by MTT assays in comparison with that of commercially available bPEI, as shown in Fig. 5. For bPEI (25 kDa) and its complexes with plasmid pMSCV-GFP-PARK2, significant cytotoxicity was observed when bPEI concentration was higher than 27.5 μg/mL and the N/P ratio was higher
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which will become an advantage when such a modified polysaccharide derivative is used for gene delivery. 3.4. Transfection effectiveness of modified amylose for plasmid pMSCV-GFPPARK2 delivery
Fig. 8. Western blot analyses of PARK2 protein expressions in 293T cells treated with plasmid pMSCV-GFP-PARK2 only (A), the modified amylose/plasmid pMSCV-GFP- PARK2 complexes (N/P = 30, B) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10, C), respectively. GAPDH was used as an internal standard for the protein loading.
than 5. In these cases, the cell viability was found to be lower than 30%. In contrast, the modified amylose and its complexes with plasmid pMSCV-GFP-PARK2 could exhibit high cell viability, even at high concentrations and N/P ratios. At the concentration of 110 μg/mL and the N/P ratio of 40, for example, the cell viability was found to be about 90% for the modified amylose and its complexes with plasmid pMSCVGFP-PARK2. Obviously, the modified amylose displayed much lower cytotoxicity when compared to bPEI, a commonly used gene vector. These results are attributed to good biocompatibity of the modified amylose,
The in vitro transfection efficiencies of modified amylose or bPEI for plasmid pMSCV-GFP-PARK2 delivery were assessed in 293T cells, as shown in Fig. 6. Depending on the N/P ratio, the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFPPARK2 complexes have various transfection efficiencies. For the modified amylose/plasmid pMSCV-GFP-PARK2 complexes, the transfection efficiency increased with the increase of N/P ratio from 10 to 30, and had a slight decrease with a further increase of N/P ratio to 40. In contrast, the transfection efficiency of bPEI/plasmid pMSCV-GFP-PARK2 complexes decreased continuously with the increase of N/P ratio from 10 to 40. A decrease of transfection efficiency at a higher N/P ratio could be attributed to the cytotoxicity [21]. In particular, an optimized N/P ratio corresponding to the maximum transfection efficiency was found to be 30 for the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and 10 for bPEI/ plasmid pMSCV-GFP-PARK2 complexes, respectively. Fig. 7 gives typical fluorescence images of 293T cells transfected by the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and bPEI/plasmid pMSCV-GFP-PARK2 complexes at various N/P ratios. As seen, the effects of N/P ratio on the fluorescence signal intensity in each case were consistent with the effects of N/P ratio on the transfection efficiency (Fig. 6). Similarly, the strongest fluorescence signal was observed at the N/P ratio of 30 for the modified amylose/plasmid pMSCV-GFP-PARK2 complexes and 10 for bPEI/plasmid pMSCV-GFPPARK2 complexes, respectively.
Fig. 9. Cell apoptosis analyses by flow cytometry for 293T cells treated with treated with plasmid pMSCV-GFP-PARK2 only (A), the modified amylose/plasmid pMSCV-GFP- PARK2 complexes (N/P = 30, B) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10, C), respectively.
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To confirm the effectiveness of modified amylose for the delivery of plasmid pMSCV-GFP-PARK2 to 293T cells, Western blot and cell apoptosis analyses were also carried out. Fig. 8 shows Western blot analyses of PARK2 protein expressions in 293T cells incubated with plasmid pMSCV-GFP-PARK2 only, the modified amylose/plasmid pMSCV-GFPPARK2 complexes (N/P = 30) and bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10), respectively. In the case of plasmid pMSCVGFP-PARK2 only, few PARK2 protein expressions were observed. In contrast, a high level of PARK2 protein expressions were found in the case of the modified amylose/plasmid pMSCV-GFP-PARK2 complexes (N/ P = 30) or bPEI/plasmid pMSCV-GFP-PARK2 complexes (N/P = 10). It is known [22] that the increase in the expression of a particular protein reported is an indirect proof of successful transfection of a specific gene. Therefore, the modified amylose has good gene delivery capability similar to commonly used bPEI gene vector. Moreover, the use of this modified amylose did not induce significant cellular apoptosis when compared to bPEI, as illustrated in Fig. 9. 4. Conclusions Although amylose has been widely used for biomedical applications, few studies have dealt with its modification for efficient gene delivery. In this work, we modified amylose by the click conjugation with propargyl focal point poly(L-lysine) dendron of third generation and then used successfully for the delivery of plasmid pMSCV-GFP-PARK2 to human embryonic kidney 293T cells. This novel amylose derivative containing poly(L-lysine) dendrons is characteristic of much lower cytotoxicity and considerable gene delivery capability when compared to branched polyethylenimine (bPEI, 25 kDa), a commonly used gene vector. Considering the supramolecular encapsulation of helical amylose to singlewall carbon nanotubes [23] or drugs [24], our next work will focus on the potential applications of such a polysaccharide conjugate for combined gene and photothermal therapy as well as gene and drug codelivery in order to enhance the therapeutic efficacy. Acknowledgments This work was supported by National Natural Science Foundation of China (51273216, 21074152, J1103305), Doctoral Research Program of Education Ministry in China (20090171110023 and 20130171120095),
Key Project of Scientific and Technical Innovation for Universities in Guangdong Province (cxzd1102) and Natural Science Foundation of Guangdong Province (S2013010012549 and S2013040014401). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.01.011. References [1] K. Raemdonck, T.F. Martens, K. Braeckmans, J. Demeester, S.C. De Smedt, Adv. Drug Deliv. Rev. 65 (2013) 1123. [2] M.D. Buschmann, A. Merzouki, M. Lavertu, M. Thibault, M. Jean, V. Darras, Adv. Drug Deliv. Rev. 65 (2013) 1234. [3] W. Khan, H. Hosseinkhani, D. Ickowicz, P.D. Hong, D.S. Yu, A.J. Domb, Acta Biomater. 8 (2012) 4224. [4] L. Casettari, D. Vllasaliu, J.K. Lam, M. Soliman, L. Illum, Biomaterials 33 (2012) 7565. [5] J.J. Thomas, M.R. Rekha, C.P. Sharma, Int. J. Pharm. 389 (2010) 195. [6] K.L. Chang, Y. Higuchi, S. Kawakami, F. Yamashita, M. Hashida, Bioconjug. Chem. 21 (2010) 1087. [7] C. Yang, H. Li, S.H. Goh, J. Li, Biomaterials 28 (2007) 3245. [8] L.B. Thomsen, J. Lichota, K.S. Kim, T. Moos, J. Control. Release 151 (2011) 45. [9] W.T. Godbey, K.K. Wu, A.G. Mikos, J. Control. Release 60 (1999) 149. [10] F.J. Verbaan, C. Oussoren, I.M. van Dam, Y. Takakura, M. Hashida, D.J. Crommelin, W.E. Hennink, G. Storm, Int. J. Pharm. 214 (2001) 99 (G.). [11] H.C. Wu, A. Sarko, Carbohydr. Res. 61 (1978) 27. [12] X. Cai, L. Yang, L.M. Zhang, Q. Wu, Carbohydr. Res. 345 (2010) 922. [13] W. Gao, B. Sha, W. Zou, X. Liang, X. Meng, H. Xu, J. Tang, D. Wu, L. Xu, H. Zhang, Biomaterials 32 (2011) 9425. [14] S.H. Moghadam, H.W. Wang, E. Saddar El-Leithy, C. Chebli, L. Cartilier, Biomed. Mater. 2 (2007) S71. [15] S. Toita, N. Morimoto, K. Akiyoshi, Biomacromolecules 11 (2010) 397. [16] S. Patnaik, K.C. Gupta, Expert Opin. Drug Deliv. 10 (2013) 215. [17] N. Pahimanolis, A.H. Vesterinen, J. Rich, J. Seppala, Carbohydr. Polym. 82 (2010) 78. [18] D. Ma, H.B. Zhang, Y.Y. Chen, J.T. Lin, L.M. Zhang, J. Colloid Interface Sci. 405 (2013) 305. [19] K. Kunath, A. von Harpe, D. Fischer, H. Petersen, U. Bickel, K. Voigt, T. Kissel, J. Control. Release 89 (2003) 113 (T). [20] H. Petersen, P.M. Fechner, A.L. Martin, K. Kunath, S. Stolnik, C.L. Roberts, D. Fischer, M.C. Davies, T. Kissel, Bioconjug. Chem. 13 (2002) 845. [21] H.L. Jiang, Y.K. Kim, R. Arote, J.W. Nah, M.H. Cho, W.J. Choi, T. Akaike, C.S. Cho, J. Control. Release 117 (2007) 273. [22] A. Bajaj, P. Kondaiah, S. Bhattacharya, J. Med. Chem. 50 (2007) 2432. [23] O.K. Kim, J. Je, J.W. Baldwin, S. Kooi, P.E. Pehrsson, L.J. Buckley, J. Am. Chem. Soc. 125 (2003) 4426. [24] B.G. Li, L.M. Zhang, Prog. Chem. 22 (2010) 1161.