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siRNA delivery vectors in vitro and in vivo
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 337 – 345
Research Article
nanomedjournal.com
Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo Sushil K. Tripathi, MSc a , Ritu Goyal, MPhil a , Pradeep Kumar, PhD a , Kailash C. Gupta, PhD a, b,⁎ a
CSIR-Institute of Genomics and Integrative Biology, Delhi University, Delhi, India b CSIR-Indian Institute of Toxicology Research, Lucknow, India Received 4 March 2011; accepted 26 June 2011
Abstract Chitosan was partially converted to its chlorohydrin derivative by the reaction with epichlohydrin, which was subsequently reacted with varying amounts of lPEI(2.5kD) to obtain a series of chitosan-lPEI(2.5kD) copolymers (CP). These copolymers were then characterized and evaluated in terms of transfection efficiency (in vitro and in vivo), cell viability, DNA release and buffering capacity. The CP-4 copolymer (the best among the CP series) showed enhanced transfection (~ 2 – 24 folds) in comparison with chitosan, lPEI(2.5kD), bPEI(25kD) and Lipofectamine in HEK293, HeLa and CHO cells. The buffering capacity (in the pH range of 3 – 7.5), as shown by confocal microscopy, and DNA-release capability of the CP copolymers, was found to be significantly enhanced over chitosan. Intravenous administration of CP-4/ DNA polyplex in mice followed by the reporter gene analysis showed the highest gene expression in spleen. Collectively, these results demonstrate the potential of CP-4 copolymer as a safe and efficient nonviral vector. From the Clinical Editor: Chitosan –PEI (2.5kD) copolymers (CP) were characterized and their transfection efficiency, DNA release and buffering capacity were studied. The CP-4 copolymer significantly enhanced buffering capacity and provided the highest gene expression levels. The method may be used to enhance DNA transfection. © 2012 Elsevier Inc. All rights reserved. Key words: Chitosan; lPEI(2.5kD); Confocal microscopy; Buffering capacity; Transfection; siRNA; In vivo gene expression
Cationic polymer-mediated gene delivery has emerged as a promising alternative to the use of viral vectors for gene therapy. 1-3 These polymers have the ability to condense DNA into a more compact form (i.e., nanoparticles [NPs]) that allow easier transport through cellular membrane and provide protection to complexed DNA against nuclease digestion. 4,5 Chitosan has shown great potential as a gene-delivery vehicle owing to its biodegradable nature and nontoxic property. 6,7 However, its extensive use in biomedical applications is restricted due to its poor transfection efficiency, which is mainly due to its i) low solubility at physiological pH, 8 ii) poor endosomal escape due to lack of buffering amines, 9 and iii) very strong binding with DNA resulting in inefficient unpackaging of genetic material in the cytoplasm. 10 The authors report no conflicts of interest. Authors gratefully acknowledge the financial support from CSIR Task Force Project (NWP035). R.G. thanks the University Grants Commission, New Delhi, India, for the award of a Senior Research Fellowship. ⁎Corresponding author: CSIR-Indian Institute of Toxicology Research, Lucknow-226001, U.P. India. CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi-110 007, India. E-mail addresses: [email protected], [email protected] (K.C. Gupta).
To address these limitations, Chang et al 11 modified chitosan by tethering histidine moieties onto the chitosan backbone and showed enhanced transfection efficiency due to the increase in buffering capacity in the pH range of endosomes and lysosomes. However, the cellular uptake was found to be lower than that of Lipofectamine (Invitrogen Inc., Carlsbad, California), a commercially available transfection reagent. Jiang et al 12 prepared chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer via condensation of periodate-oxidized chitosan and bPEI(1.8kD) followed by reduction of the resulting Schiff's bases. They demonstrated comparable transfection efficiency of the grafted polymer with that observed with Lipofectamine. A number of other modifications, such as trimethylation, 13 PEGylation, 14,15 depolymerization 16,17 and galactosylation, 18 have also improved the transfection efficiency of chitosan with limited success. Alternatively, in an elegant fashion, Chen et al 19,20 have employed a dissimilar approach in exploring the transport mechanism of PEG-modified polylysine compacted DNA NPs using lipid raft-mediated endocytosis to enter the cells. We, therefore, proposed to better the transfection efficiency of chitosan well above the reported success by introducing a
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.06.022 Please cite this article as: S.K. Tripathi, R. Goyal, P. Kumar, K.C. Gupta, Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo. Nanomedicine: NBM 2012;8:337-345, doi:10.1016/j.nano.2011.06.022
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cationic polymer onto the chitosan backbone using a novel approach. The projected grafting circumvents all the limitations (solubility, buffering and DNA binding properties) and simultaneously maintains the integrity of the chitosan backbone. For this purpose, we deliberately selected a low molecular weight lPEI(2.5kD), which possesses an adequate amount of buffering capacity but is a poor transfection reagent. 21,22 In this study, we prepared chitosan- lPEI(2.5kD) (CP) copolymers by partially converting chitosan hydrochloride into its chlorohydrin, which further partially reacts with 2° amines of lPEI(2.5kD) to convert them into 3° by keeping the total number of amines intact in the resulting copolymer. By doing so, the lPEI(2.5kD) grafted copolymer then had all types of amines (1°, 2°, 3°) that are required for good buffering, proper binding and release of DNA. 23-25 Adopting this strategy, a small series of graft copolymers (CP-1 to CP-5) was prepared by varying the amount of lPEI(2.5kD). The resulting CP copolymers, soluble at physiological pH, were subsequently characterized spectroscopically and evaluated in terms of their buffering capacity, DNA binding and release capability, the ability to carry nucleic acids in vitro and in vivo, and cytotoxicity. 26 One of the graft copolymers among the series, CP-4, demonstrated ~ 2 – 24-fold higher gene delivery ability in comparison with native chitosan, lPEI(2.5kD), bPEI(25kD) and selected commercial transfection reagents, such as Superfect (Qiagen SAS, Courtaboeuf, France) and Lipofectamine.
Methods Preparation of chitosan chlorohydrin (CC) Chitosan (100 mg, 1 mg/ml in 1% aqueous HCl) was dissolved by keeping it overnight on stirring. The resulting chitosan hydrochloride solution (100 ml) was stirred at 65°C and to it added epichlorohydrin (0.25ml). Stirring was continued for ~ 20 h at the same temperature and then concentrated to dryness on a rotary evaporator to obtained chitosan chlorohydrin hydrochloride (CC) in ~ 95% yield as a white powder. Preparation of CP CC (25 mg) and lPEI(2.5kD) (25 mg) were suspended in water (25ml, 1mg/ml) and heated to 65°C. An aqueous solution of sodium hydroxide (2 N, 2 ml) was added dropwise to the above rapidly stirred reaction mixture. The reaction was allowed to stir for ~ 20h at 65°C. Thereafter, the resulting solution was concentrated on a rotary evaporator and dialyzed against water for two days with intermittent change of water. The dialyzed solution was lyophilized in a speed vac to obtain CP-1 copolymer in ~ 85% yield. Similarly, other copolymers with increasing amounts (weight ratio) of lPEI(2.5kD), viz., 1:2 (CP-2), 1:3 (CP3), 1:4 (CP-4) and 1:5 (CP-5) were prepared in ~ 78 – 85% yield and characterized by 1H-NMR (DRX-300, 300 MHz, FTNMR, Bruker Avance, Madison, Wisconsin). Buffering capacity Chitosan, bPEI(25kD), lPEI(2.5kD) and CP copolymers (3 mg each, separately) were dissolved in 0.1 N NaCl to obtain
concentrations of 0.1 mg/ml. Aqueous HCl (0.1 M) and NaOH (0.1 M) were used as titrators. The buffering capacity of each of the polycations was determined using pH titration from low to high. Endosomal escape through confocal microscopy Endosomal escape of CP-4 and chitosan was monitored on HeLa cells. Endosomes were labeled with Alexa Fluor 488 (100 μg/ml) and lysosomes with Lysosensor Green DND-189 (1 μM) and Lysotracker Green DND-26 (1 μM). These antibodies were added to the cells prior to addition of the chitosan and CP-4 complexes containing tetramethylrhodamine-labeled pDNA, and incubated for 20 minutes in OptiMEM I medium. Subsequent to the addition of the complexes, the cells were incubated for 1h and then washed with 1 × phosphate buffered saline (PBS) (2 × 1 ml) followed by fixing with 4% paraformaldehyde. The images were recorded using a ZEISS LSM 510 inverted microscope. In vitro transfection GFP plasmid transfection To evaluate the gene-carrying efficiency of CP copolymers, HEK293, CHO and HeLa cells were seeded 1 day prior to transfection assay in 96-well plates after which media was aspirated and cells were washed with PBS (1 × PBS). CP copolymers/bPEI(25kD)/lPEI(2.5kD)/chitosan were complexed with pDNA (0.3 μg) at w:w ratios of 1:1, 1.66:1, 2.33:1, 3.3:1, 4:1 and 5:1. Similarly, pDNA complexes were prepared with the standard transfection reagents, viz., Superfect, Fugene (Roche Inc., Branchburg, New Jersey), GenePORTER 2 (Genlantis Inc., San Diego, California) and Lipofectamine, following manufacturers' protocols. DNA complexes were diluted with serum-free DMEM (60 μl) and gently added to HEK293, CHO and HeLa cells. In another set, DNA complexes were diluted in DMEM supplemented with 10% FBS and added to various cells. After 4 h, the transfection medium was replaced by 200 μl fresh DMEM containing 10% FBS and cells were further incubated for 36 h. EGFP expression in mammalian cells was quantitatively estimated on NanoDrop ND- 3000 spectrofluorometer (Thermo Scientific, Wilmington, Delaware) after 36 hours. Similarly, for FACS analysis, HEK cells were seeded in 24well plates 1 day prior to transfection and the analysis was performed 36 h post transfection. CLSM Intracellular trafficking studies of CP-4/DNA and chitosan/ DNA complexes were performed on HeLa cells. For this, pDNA was labeled with YOYO-1 iodide; CP-4 and chitosan were labeled with tetramethylrhodamine isothiocyanate (TRITC). Briefly, YOYO-1 iodide (2 μl, 1 mM solution in DMSO) was added to pDNA (0.3 mg) and the resulting mixture was kept stirring for 2h at 25 ± 1°C in dark conditions and finally stored at -20°C. CP-4 and chitosan (10 mg/ml) were treated overnight with TRITC (26 μl, 1 mg/100 μl DMF) for 1% blockage of total amines in polymers. Subsequently, the reaction mass was concentrated in a speed vac and unreacted/hydrolyzed TRITC was removed by trituration with ethyl acetate (3 × 2ml). The resulting product was completely dried and resuspended in water.
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Table 1 Particle size and zeta potential measurements of CP/DNA nanoplexes in water and serum at w/w ratio of 2.33:1 and lPEI(2.5kD)/DNA and chitosan/DNA complexes at 1.66:1. PDI = 0.15 – 0.34 S. No.
1. 2. 3. 4. 5. 6. 7.
Sample ID
CP-1 CP-2 CP-3 CP-4 CP-5 Chitosan lPEI (2.5kD)
Average particle size in nm ± SD
Zeta potential in mV ± SD
DNA-loaded copolymer (in H2O)
DNA-loaded copolymer (in DMEM)
Copolymer (in H2O)
DNA-loaded copolymer (in H2O)
DNA-loaded copolymer (in DMEM)
100 ± 1.52 90.4 ± 1.56 85 ± 3.68 75.6 ± 3.26 65.5 ± 3.52 136.3 ± 5.38 128.7 ± 4.21
85 ± 7.2 75 ± 6.4 68 ± 1.5 60 ± 3.7 55 ± 4.4 60 ± 4.3 61 ± 7.0
44.7 ± 0.87 48.3 ± 4.82 50.3 ± 0.96 53.2 ± 2.96 56 ± 1.73 30.5 ± 2.0 25.7 ± 1.17
39.7 ± 1.3 42.3 ± 1.8 45.7 ± 1.3 44.1 ± 1.6 50.8 ± 1.4 26.8 ± 2.1 22.1 ± 1.4
-11.56 ± 2.2 -10.23 ± 1.6 -9.38 ± 1.3 -11.86 ± 1.58 -10.76 ± 1.69 -9.48 ± 1.48 -9.78 ± 1.36
HeLa cells were seeded (1.5 × 10 5 cells/well) on circular glass coverslips in 6-well plates, grown overnight to ~ 70% confluence. A polyplex solution (500 μl) containing DNA (0.3 μg) in DMEM was added to each well. After incubation for 30, 60 and 120 minutes, the cells were washed with 1 × PBS (3 × 1 ml) and fixed with 4% paraformaldehyde solution for 10 minutes at 37°C. The fixed cells were counter-stained with a blue nuclear dye, DAPI, and the coverslips were mounted on glass slides with fluorescence-free Mounting Medium (UltraCruz, Santa Cruz Biotechnology, Santa Cruz, California). All confocal images were acquired using a Zeiss LSM 510 inverted confocal laser-scanning microscope (CLSM). Endosomal and lysosomal escape of complexes was also monitored by confocal microscopy. The detailed experimental procedure is given in the Supplementary Information found in the online version of this article.
Weight ratio of copolymer/DNA complexes
2.33:1 2.33:1 2.33:1 2.33:1 2.33:1 1.66:1 1.66:1
Results
chlorohydrine and CP copolymers were synthesized, as described above and depicted in Scheme-S1 (see Supplementary Information), in ~ 95 and ~ 78 – 85% yield, respectively. The degree of grafting of chlorohydrin followed by lPEI(2.5kD) on synthesized copolymers was determined by 1H-NMR spectroscopy. From integration of signals, the substitution of 3-chloro-2-hydroxypropyl groups onto a chitosan backbone was found to be 45%, i.e., ~ every two units of glucosamine contain one 3-chloro-2-hydroxypropyl group. Further, the percent substitution of linear PEI onto chitosan chlorohydrin is shown in Table S1 (see Supporting Information). The size and surface charge of the particles formed with DNA was measured by DLS and zeta sizer. The size of CP-1 to CP-5 and their pDNA complexes was measured in water and 10% serum at a weight ratio of 2.33:1 (copolymer:DNA) for the CP series, 1.66:1 for chitosan and lPEI(2.5kD). In water, moving down the series from CP-1 to CP-5, size of the particles was found to be inversely proportional with the concentration of lPEI(2.5kD) (data not shown). The same trend was observed on complexation with pDNA, though a further decrease in size of the nanoplexes may be due to the formation of more compact complexes (Table 1). In presence of serum, an additional decrease in the size of the particles was observed from CP-1 to CP-5. Unlike size, the zeta potential of the particles increased upon increasing the amount of lPEI(2.5kD) (ranging from ~ +44.7 - +56mV) in the copolymers. However, the zeta potential of particles decreased after complexation with pDNA and it became negative in presence of serum. To establish the pDNA binding ability of the copolymers, a gel retardation assay was carried out at different weight ratios. The binding of CP copolymers to DNA neutralizes the negative charge in the phosphate backbone, which resulted in the decreased mobility of DNA under the influence of electrical current. We observed that on increasing the amount of lPEI (2.5kD) in CP copolymers (CP-1 to CP-5), the electrophoretic mobility of DNA decreased with lesser amounts of copolymers and at a copolymer:DNA ratio of 1:1 (w:w) in the case of CP-4 and 5, the mobility of pDNA was completely retarded (Figure S1, Supporting Information). lPEI(2.5kD) also retarded the DNA mobility at the same weight ratio.
Preparation and characterization of CP copolymers
Buffering capacity and endosomal release
Chitosan-lPEI(2.5kD) copolymers (CP-1 to CP-5) were prepared by increasing the amount of lPEI(2.5kD). Chitosan
The endosomal release of DNA nanoplexes in the cytoplasm is one of the important parameters that depends on the intrinsic
In vivo gene expression in Balb/c mice pGL3 (25μg) was complexed with CP-4 at a w:w ratio of 1:2.33 and bPEI(25kD) at 1:1.66, and the final volume was made up to 100 μl using normal saline. The complexes were incubated for 30 min at 25 ± 1°C and injected into 3 Balb/c mice through the tail vein using an insulin syringe (1 ml) with needle size of 0.30 × 8mm. The animals were kept for 7 days on a normal diet, after which they were sacrificed by cervical dislocation and all the vital organs were dissected out. The organs were washed with chilled normal saline, weighed, chopped and made 25% w/v homogenate in 1 × cell lysis buffer (Promega Corp., Madison, Wisconsin) containing 1 × protease inhibitor cocktail. After 3 cycles of freezing and thawing in liquid nitrogen and at 37°C, respectively, the whole homogenate was centrifuged at 10,000 rpm for 10 minutes at 4°C. Cell lysate (100 μl) from each sample was assayed for luciferase activity by adding 100 μl luciferin substrate on the luminometer (Berthold Technologies, Pforzheim, Germany). The standard curve was drawn using luciferase enzyme provided with the kit. The same experiment was repeated twice.
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Figure 1. (A) Buffering capacity of lPEI(2.5kD), chitosan and CP copolymers. (B) Percent transfection efficiency in the presence of Bafilomycin A1 (Proton pump inhibitor) of bPEI(25kD), chitosan and CP-4 copolymer.
buffering capacity of the vector system, which is measured by acid-base titration. The standard acid-base titration of lPEI (2.5kD) showed a considerable buffering capacity over a pH range of 3 to 10; however, due to limited solubility of CP copolymers at higher pH, the titration was carried out over a pH range of 3 to 7.5. The volume of NaOH required for bringing the pH from 3 to 7.5 in the case of lPEI(2.5kD) and bPEI(25kD) was significantly higher (P b 0.05) than the volume required for chitosan (Figure 1, A). It is interesting to note that for CP copolymers, the volume of NaOH required was comparable with that of lPEI(2.5kD) and bPEI(25kD). Effect of H +-ATPase inhibitor The effect of Bafilomycin A1 (a proton pump inhibitor) was observed on the transfection efficiency of CP-4/DNA, bPEI (25kD)/DNA and chitosan/DNA on HEK293 cells using FACS. A considerable decrease in transfection efficiency was noticed in the case of CP-4 (~ 47% ca. ~ 8% with Bafilomycin A1) and bPEI(25kD) (~ 25% ca. ~ 6% with Bafilomycin A1), whereas the decrease in the case of chitosan (~ 2% ca. ~ 1.6% with Bafilomycin A1) was insignificant (Figure 1, B). Toxicity assessment The cytotoxicity of CP copolymers /DNA complexes at different weight ratios was investigated using MTT assay on HEK293, HeLa and CHO cells and compared with lPEI(2.5kD), bPEI(25kD), chitosan and the commercial transfection reagents. In that cytotoxicity investigation, bPEI(25kD) showed significant cytotoxicity (P b 0.05) in comparison with lPEI(2.5kD), chitosan and CP copolymers. However, lPEI(2.5kD) showed minimal cytotoxicity at its best working concentration on all the cell lines (Figure 2). Chitosan and CP copolymers did not show any cytotoxicity as evidenced by the healthy growth of the cells with more than 100% cell viability. Transfection efficiency measurements of CP copolymers Using a plasmid containing enhanced green fluorescent protein (EGFP) as the reporter gene, we compared the
Figure 2. Cell viability profile of CP/DNA nanoplexes at an w/w ratio of 1:2.33, lPEI(2.5kD)/DNA and chitosan/DNA polyplex at w/w ratio of 1:1.66, and Superfect/DNA, Fugene/DNA, GenePORTER 2/DNA and Lipofectamine/DNA complexes in HEK293, HeLa and CHO cells. Cells were treated with DNA complexes under conditions described under transfection assay, and cytotoxicity was determined by MTT assay. Percent viability of cells is expressed relative to control cells. Each point represents the mean of 3 independent experiments performed in triplicate. **P b 0.01 vs. PEI and Lipofectamine in all the cell lines.
transfection efficiency of CP copolymers with those of lPEI (2.5kD), bPEI(25kD), chitosan and selected commercial transfection reagents, viz., Superfect, Fugene, GenePORTER 2 and Lipofectamine. The same w:w ratio of copolymer:DNA (see gel retardation assay) was used in all the transfection experiments. Thirty-six hours post transfection, EGFP fluorescence was observed. Following spectrofluorometric quantification, transfection efficiency of CP copolymers at w:w ratio 2.33 was found to be significantly higher (P b 0.05) than that of lPEI(2.5kD), bPEI(25kD) and chitosan at w:w ratio of 1.66 (Figure S2, Supporting Information) in the presence and absence of serum. It is interesting to note that CP-4 among the series of synthesized copolymers displayed ~ 20- and 24-fold (P b 0.05) GFP expression in HEK293 cells in comparison with chitosan and lPEI(2.5kD), respectively, in both the presence or absence of serum. Further, with CP-4, EGFP expression was ~ 1.3- to 3.0fold higher (P b 0.05) in this particular cell line in comparison with bPEI(25kD), GenePORTER 2, Superfect, Fugene and Lipofectamine in both the presence or absence of serum (Figure S2, Supporting Information). A similar trend was observed in the other cells, viz., HeLa and CHO. To find out the cell population expressing GFP, FACS analysis was performed with the cells transfected with copolymer:DNA over a range of w:w ratios. We observed a maximum percentage (~ 45%) of GFP positive cells transfected with CP-4 at a w:w ratio of 1:2.33; however, GFP expression regressed with a deviation from the above ratio (Figure 3). In contrast, chitosan and lPEI(2.5kD) showed only ~ 3 and ~ 2% GFP positive cells, respectively, at a w:w ratio of 1:1.66. Although only 26% GFP positive cells were scored after transfection with bPEI(25kD), a gold standard for the transfection studies, at a w:w ratio of 1:1.66; 16% and 35% GFP positive cells were observed following transfection with selected commercial reagents, viz., Lipofectamine and Superfect, respectively.
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Figure 3. Percent transfection efficiencies of CP-3, CP-4, and CP-5/DNA nanoplexes determined using FACS at various w/w ratios and compared with lPEI(2.5kD), chitosan, Superfect and Lipofectamine. **P b 0.01 vs. PEI (at all w:w ratios), Superfect and Lipofectamine.
siRNA Delivery efficiency The delivery of two types of siRNAs, viz., GFP specific and JNKII, was tested on HEK293 cells. The transfection was performed in a sequential manner using GFP/CP-4 system, as described above. The observed knockdown of GFP expression by CP-4/siRNA system was found to be ~ 77%, whereas only 49% knockdown was recorded with Fugene/siRNA system using nanodrop spectrofluorometry (Figure 4, A). As a proof of principle, we tested another siRNA, JNKII, which is activated by a variety of environmental stresses. 30 Using western hybridization, we observed nearly 60% knockdown of JNKII using CP-4 as a transfection agent after 36 hours of treatment, which was comparable with that observed with the commercial transfection reagent, Lipofectamine (~ 55%) (Figure 4, B). This further boosts the potential of CP-4 as a versatile gene-delivery agent. pDNA binding affinity The binding ability of CP-4, chitosan and lPEI(2.5kD) with pDNA was studied by incubating the polyplex solution with increasing amounts of heparin, a competitive anionic moiety, and the subsequent release pattern of pDNA was recorded on gel electrophoresis. As expected, only ~ 30% of bound DNA was found to be released from chitosan, even after addition of 35 U of heparin, whereas ~ 96% DNA was released from lPEI(2.5kD) in the presence of 2.5 U heparin and ~ 72% DNA was released from bPEI(25kD) with 5 U heparin. It is interesting to note that we observed ~ 92% of DNA released from CP-4 copolymer in the presence of 5 U of heparin (Figure S3, Supporting Information).
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Figure 4. (A) CP-4 copolymer was checked for its ability to deliver siRNA in HEK293 cells by transfecting pEGFP DNA followed by GFP specific siRNA. The expression of GFP in cells was suppressed by ~ 77%, in comparison with Fugene/siRNA, which suppressed GFP expression by only ~ 49%, as monitored by measuring fluorescence on a spectrofluorometer. All the experiments were performed at least thrice and error bars represent the SD, (B) Immunoblotting assay for CP-4 to see the JNKII knockdown. Lane 1: CP-4/JNKII siRNA, lane 2: CP-4 without siRNA, Lane 3: Lipofectamine without siRNA, Lane 4: Lipofectamine/JNKII siRNA.
complexed with CP-4 copolymer did not show any significant degradation (~ 15% after 2h). Intracellular trafficking To investigate the path of cellular entry, tetramethylrhodamine-labeled CP-4 was complexed with YOYO-1-labeled pDNA and added on to HeLa cells, which were fixed with paraformaldehyde at specified time intervals. We observed that CP-4 was able to carry pDNA to the insides of the cells (cytoplasm) within 30 minutes of the addition to the cells. Further, both pDNA and CP-4 were observed inside the nucleus after 1h of the addition of the complexes, and after 2 h, the maximum amount of fluorescence was observed inside the nucleus only (Figure 5). HeLa cells, prelabeled with endosomal and lysosomal dyes (green), were incubated with tetramethylrhodamine (TMR) labeled pDNA/chitosan and CP-4 complexes (red). After imaging through confocal microscopy, it was observed that in case of chitosan, red and green regions were overlapping, showing the entrapment of these complexes within these compartments (Figure S5, Supporting Information), whereas, in the case of CP-4, separate red and green regions were observed, indicating the release of these complexes from these compartments.
Protection of pDNA against nucleases Having evaluated the binding and release of DNA from the copolymer, CP-4, it becomes mandatory to examine the integrity of pDNA bound to copolymer against nucleases. DNase I protection assay was performed and the results analyzed by 0.8% agarose gel electrophoresis. Figure S4 (Supporting Information) clearly depicts that whereas naked DNA (0.3 μg) was found to be degraded within 15 minutes of incubation with DNase I, pDNA
Protein adsorption assay The amount of protein adsorbed onto the surface of CP-4 was evaluated on SDS-PAGE taking BSA as a standard. We observed significantly reduced binding of BSA with our test copolymer, CP-4, as evidenced in Figure S6 (Supporting Information), indicating a reduction in non-specific binding of CP-4 to serum proteins.
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Figure 5. Confocal microscopic images of HeLa cells treated with tetramethylrhodamine-CP-4/YOYO-1-pDNA nanoplex at different time points.
In vivo gene expression in Balb/c mice Having achieved the significant level of transfection in vitro, we further examined the efficacy of CP-4 for its in vivo applications and used a firefly luciferase expression-reporter system in Balb/c mice. We observed a maximum luciferase activity in spleen (P b 0.05) followed by heart and brain in the mice after 7 days in comparison with bPEI(25kD) (Figure 6). We did not use chitosan and lPEI(2.5kD) in this study because none of those showed any significant transfection in vitro. Discussion Figure 6. In vivo gene expression analysis in Balb/c mice 7 days post i.v. injection using pGL3 control vector as a reporter gene. The mice were sacrificed and various organs such as heart, liver, spleen, kidney, brain and lungs were dissected out. The organs were homogenized in lysis buffer and the lysate was read on luminometer to quantify the luciferase activity. *P b 0.05 vs. PEI in the respective organs.
In this study, we tried to improve the transfection efficiency of chitosan and while designing the strategy for the preparation of modified chitosan, we were guided by four important considerations, viz., (i) the modified chitosan should be soluble at physiological pH, (ii) it should have improved buffering capacity so that desired therapeutics is released in cytoplasm, (iii) it should efficiently release DNA in the cytoplasm for nuclear
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localization, and (iv) the modified analogue of chitosan should retain the property of chitosan with respect to biocompatibility and cell viability. PEIs are known to possess very good buffering capacity; however, they exhibit molecular weight-dependent transfection efficiency. Branched PEIs with high molecular weight have better gene transfer capability but suffer from chargeassociated toxicity. PEIs with low molecular weight are nontoxic but offer poor transfection efficiency. To take advantage of the stated properties of low molecular weight PEIs and to satisfy all the above criteria, we deliberately selected low molecular weight cationic polymers (bPEI1.8kD and lPEI2.5kD) for grafting on chitosan using a two-step methodology. In the first step, the chitosan hydrochloride was converted into its chlorohydrin derivative by the reaction with epichlorohydrin, which was subsequently reacted with 1° and 2° amines of bPEI(1.8kD) via in situ generation of epoxide under alkaline conditions followed by the opening of the epoxide ring to prepare a series of bPEI(1.8kD)-graft-chitosan copolymers by varying the amount of bPEI(1.8kD). The resulting copolymers were tested for their in vitro transfection efficiency and found to be incomparable with bPEI(25kD) (a gold standard) (data not shown). Subsequently, we prepared another series of graft-copolymers by replacing bPEI(1.8kD) with lPEI(2.5kD) in an analogous manner and made a series of graft-copolymers by varying the amount of lPEI(2.5kD) (Scheme S1, Supporting Information). The percentage of grafting was determined by 1H-NMR, which confirmed that on increasing the concentration of lPEI(2.5kD), down the series, there is a gradual increase in the substitution of lPEI (2.5kD) on the chitosan backbone (Table S1, Supporting Information). All the copolymers in the series were evaluated in terms of cell viability and transfection efficiency in a variety of cell lines. The transfection efficiency of this series was found to be dramatically improved, i.e., 2- to 24-fold higher than those of chitosan, lPEI(2.5kD), bPEI(25kD) and Lipofectamine. We, therefore, proceeded with linear chitosan-lPEI (2.5kD) copolymers for subsequent studies and evaluated them for buffering capacity, DNA release and in vitro and in vivo gene expression. The size and surface charge of NPs are the vital parameters to determine their cellular uptake and interactability with the membrane. In the proposed strategy, the copolymer resulting from the mixing of two polymers should exhibit a decrease in size (due to increasing cross-linking leading to compactness) and an increase in zeta potential (due to incorporation of the more positive charge of PEI) on increasing the percent cross-linking of PEI in proceeding down the series from CP-1 to CP-5. As expected, the size of DNA complexes of CP copolymers in the presence of water and serum was found to decrease on descending the series. Such a decrease in the size of the complex in water might be due to the greater compactness of complexes on increasing the amount of PEI in the series. In presence of serum, the further decrease might be due to the adsorption of water from the complexes by the negatively charged serum proteins. Additionally, the increase in zeta potential on descending the series further substantiated the attachment of lPEI(2.5kD) with chitosan.
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The high transfection efficacy of PEI polyplexes is attributed to their excellent buffering capacity in the endosomes. 27 Conversely, due to the poor buffering capacity of chitosan, their polyplexes are unable to rupture the endosomes and are degraded into lysosomes, resulting in poor transfection efficiency. 21 On the other hand, CP copolymers were found to possess improved buffering capacities (comparable with that of PEIs) and these observations were indicative of improved transfection efficiency of CP copolymers over lPEI(2.5kD), bPEI(25kD) and chitosan. Further, to confirm the above observation, transfection assay was carried out in the presence of Bafilomycin A1 (a proton pump inhibitor), which resulted in the substantial decrease in the endosomal mediated transfection efficiency of PEI (~76%). 26 Similar results were obtained with CP-4 formulation (~ 83% decrease in the transfection efficiency); however, insignificant change in transfection efficiency was observed with the chitosan formulation. The results indicated that CP-4, having comparable buffering capacity to branched PEI(25kD), followed pathways identical to PEI, whereas chitosan, which does not possess any buffering capacity, could not rupture the endosomes. The buffering capacity was also evaluated through confocal microscopy for the CP-4 and chitosan complexes. 11 The merged green and red regions (Figure S5, Supporting Information), in the case of chitosan, showed that these complexes could not escape the endosomes and lysosomes, whereas, in the case of CP-4/ DNA complex, independent scattered red and green areas showed that these particles came out of endosomes efficiently supporting the above experiment. Prior to transfection assays, it is pertinent to test the cytotoxicity of the projected copolymers. The CP copolymers were found to possess almost negligible toxicity in comparison with bPEI(25kD) in all the cell lines tested. Because both the polymers used in this study are by themselves nontoxic to the cells, the resulting copolymers also exhibited a similar noncytotoxic effect. In addition, bPEI(25kD) was found to be toxic to the cells as it is also reported that the primary amino groups present in bPEI(25kD) cause cytotoxicity through perturbation of protein kinase activity. 28 Having confirmed the non-cytotoxic nature of the CP copolymers, we evaluated the gene transfer ability of these copolymers on a variety of cells, viz., HEK293, CHO and HeLa cells. The enhanced transfection efficiency of CP copolymers might be due to increased solubility (~ 10 mg/ml, data not shown) at physiological pH and improved buffering capacity due to lPEI (2.5kD) grafts. We also did not observe any significant change (P N .05) in transfection efficiency of CP-4 copolymers in the presence or absence of serum. This finding was further supported by an observation wherein little or negligible quantity of BSA was adsorbed onto the surface of CP-4 in comparison with lPEI (2.5kD) and chitosan (see protein adsorption assay). Therefore, diminishing interaction between serum and CP-4 could be due to the presence of hydrophilic chitosan, a sugar moiety, known for its properties to reduce interactions with serum proteins. 29 We further observed that transfection efficiency increased with the increase in the weight ratio of lPEI(2.5kD), reached up to a maximum for CP-4 and thereafter started decreasing, indicating
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CP-4 as the best carrier among the series of synthesized copolymers. Thus, in this study, the w:w ratio was found to have an important impact on the transfection ability of the synthesized copolymers and CP-4, among the tested transfection agents, emerged as the best formulation. We also established the versatility of the test copolymer, CP-4, as an efficient carrier for nucleic acids using two types of siRNAs, viz., GFP specific and JNKII on HEK 293 cells. CP-4 was able to deliver both, as could be seen by the suppression in the expression of targeted genes. To achieve successful transfection, it is a prerequisite that the bound pDNA must be released from the polyplex. The extent of binding of DNA to the cationic polymer is one of the major factors influencing transfection efficiency. Chitosan has been reported to interact with pDNA via strong electrostatic/hydrogen bonding or via hydrophobic interactions. 11 The interactions between chitosan and pDNA are quite strong and are not affected even at very high salt/detergent concentration as well as in the presence of high concentrations of heparin. 30 The binding ability was found to be in the order Chitosan⋙bPEI(25kD)NCP4NlPEI(2.5kD). These observations suggest that binding capability of CP copolymer was neither strong nor loose, which may further account for its high transfection efficiency. Sugars are known to reduce the nonspecific interactions with blood serum proteins and increase the blood circulation time. 29 To validate the same observation, the amount of protein adsorbed on to the surface of CP-4 was evaluated on SDSPAGE taking BSA as a standard. It is interesting to note that CP-4 displayed much reduced interactions with serum proteins in comparison with PEI. Sufficient protection of bound DNA from DNase I up to 2 h and reduced binding with serum proteins (BSA) suggests that the synthesized copolymer, CP-4, has the potential to be used for in vivo administration of nucleic acids. After successful transfection was achieved by CP copolymers, it was rationalized to track the path of cellular entry of DNA through confocal microscopy. The observations clearly state that the CP copolymers are capable of efficiently carrying the desired gene inside the cells. The higher uptake and transfection efficiency of CP-4 might be due to presence of sugar moieties, which helped in reducing the interactions with serum proteins present in the blood and, hence, prolonged the circulation time in the blood. 31 Luciferase activity was observed significantly in spleen, brain and heart. Spleen and liver have irregular fenestrations and also endothelia with large meshes, which allow extravasation of molecules up to 0.1 – 1 μm. 32 It was surprising that, in our experiments, negligible luciferase activity was observed in the liver and at present, we do not have any plausible explanation for that circumstance. The encouraging results of improved gene expression in the brain may be useful for brain targeting but increased gene expression in spleen indicated higher clearance of the vector by this organ and as a result shortened gene expression in the other organs. Although several reports are available on the expression and tissue distribution of the test gene(s), 17,33,34 little is known about the mechanism of distribution. Future studies would be undertaken in our laboratory to enhance understanding of the mechanism and the explanation for it. The results
presented here may be useful for the biologists working in the same field. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.nano.2011.06.022.
References 1. Lehrman S. Virus treatment questioned after gene therapy death. Nature 1999;401:517-8. 2. Liu Q, Muruve DA. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 2003;10:935-40. 3. Lollo CP, Banaszczyk MG, Chiou HC. Obstacles and advances in nonviral gene delivery. Curr Opin Mol Ther 2000;2:136-42. 4. Davis ME. Non-viral gene delivery system. Curr Opin Biotechnol 2002;13:128-31. 5. Kaneda Y, Amiji MM. Polymeric gene delivery: principles and applications. Boca Raton, Florida: CRC Press LLC, Ed. 2005. 6. Mumper RJ, Wang J, Claspell JM, Rolland AP. Novel polymeric condensing carriers for gene elivery. Proc Int Symp Control Release Bioact Mater 1995;22:178-9. 7. Borchard G. Chitosans for gene delivery. Adv Drug Deliv Rev 2001;52:145-50. 8. Aiba S. Studies on chitosan: 2. Solution stability and reactivity of partially N-acetylated chitosan derivatives in aqueous media. Int J Biol Macromol 1989;11:249-52. 9. Wong K, Sun G, Zhang X, Dai H, Liu Y, He C, et al. PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo. Bioconjug Chem 2006;17:152-8. 10. Lavertu M, Methot S, Tran-Khanh N, Buschmann MD. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation. Biomaterials 2006;27:4815-24. 11. Chang KL, Higuchi Y, Kawakami S, Yamashita F, Hashida M. Efficient gene transfection by histidine-modified chitosan through enhancement of endosomal escape. Bioconjug Chem 2010;21:1087-95. 12. Jiang HL, Kim YK, Arote R, Nah JW, Cho MH, Choi YJ, et al. Chitosangraft-polyethylenimine as a gene carrier. J Control Release 2007;117: 273-80. 13. Germershaus O, Mao S, Sitterberg J, Bakowsky U, Kissel T. Gene delivery using chitosan, trimethylchitosan or polyethylenglycol-grafttrimethylchitosan block copolymers: establishment of structure–activity relationships in vitro. J Control Release 2008;125:145-54. 14. Case AH, Picola IPD, Zaniquelli MED, Fernandes JC, Taboga SR, Winnik FM, et al. Physicochemical characterization of nanoparticles formed between DNA and phosphorylcholine substituted chitosans. J Coll Interface Sci 2009;336:125-33. 15. Xu Z, Wan X, Zhang W, Wang Z, Peng R, Tao F, et al. Synthesis of biodegradable polycationic methoxy poly (ethylene glycol)–polyethylenimine–chitosan and its potential as gene carrier. Carbohydr Polym 2009;78:46-53. 16. Morris VB, Neethu S, Abraham ET, Pillai CKS, Sharma CP. Studies on the condensation of depolymerized chitosans with DNA for preparing chitosan–DNA nanoparticles for gene delivery applications. J Biomed Mater Res Part B - App Biomater 2008;89:282-92. 17. Maclaughlin LFC, Mumper RJ, Wang J, Tagliaferri JM, Gill I, Hinchcliffe M, et al. Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. J Control Release 1998;56:259-72. 18. Song B, Zhang W, Peng R, Huang J, Nie T, Li Y, et al. Synthesis and cell activity of novel galactosylated chitosan as a gene carrier. Colloids Surf 2009;70:181-6.
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Research Article
nanomedjournal.com
Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo Sushil K. Tripathi, MSc a , Ritu Goyal, MPhil a , Pradeep Kumar, PhD a , Kailash C. Gupta, PhD a, b,⁎ a
CSIR-Institute of Genomics and Integrative Biology, Delhi University, Delhi, India b CSIR-Indian Institute of Toxicology Research, Lucknow, India Received 4 March 2011; accepted 26 June 2011
Abstract Chitosan was partially converted to its chlorohydrin derivative by the reaction with epichlohydrin, which was subsequently reacted with varying amounts of lPEI(2.5kD) to obtain a series of chitosan-lPEI(2.5kD) copolymers (CP). These copolymers were then characterized and evaluated in terms of transfection efficiency (in vitro and in vivo), cell viability, DNA release and buffering capacity. The CP-4 copolymer (the best among the CP series) showed enhanced transfection (~ 2 – 24 folds) in comparison with chitosan, lPEI(2.5kD), bPEI(25kD) and Lipofectamine in HEK293, HeLa and CHO cells. The buffering capacity (in the pH range of 3 – 7.5), as shown by confocal microscopy, and DNA-release capability of the CP copolymers, was found to be significantly enhanced over chitosan. Intravenous administration of CP-4/ DNA polyplex in mice followed by the reporter gene analysis showed the highest gene expression in spleen. Collectively, these results demonstrate the potential of CP-4 copolymer as a safe and efficient nonviral vector. From the Clinical Editor: Chitosan –PEI (2.5kD) copolymers (CP) were characterized and their transfection efficiency, DNA release and buffering capacity were studied. The CP-4 copolymer significantly enhanced buffering capacity and provided the highest gene expression levels. The method may be used to enhance DNA transfection. © 2012 Elsevier Inc. All rights reserved. Key words: Chitosan; lPEI(2.5kD); Confocal microscopy; Buffering capacity; Transfection; siRNA; In vivo gene expression
Cationic polymer-mediated gene delivery has emerged as a promising alternative to the use of viral vectors for gene therapy. 1-3 These polymers have the ability to condense DNA into a more compact form (i.e., nanoparticles [NPs]) that allow easier transport through cellular membrane and provide protection to complexed DNA against nuclease digestion. 4,5 Chitosan has shown great potential as a gene-delivery vehicle owing to its biodegradable nature and nontoxic property. 6,7 However, its extensive use in biomedical applications is restricted due to its poor transfection efficiency, which is mainly due to its i) low solubility at physiological pH, 8 ii) poor endosomal escape due to lack of buffering amines, 9 and iii) very strong binding with DNA resulting in inefficient unpackaging of genetic material in the cytoplasm. 10 The authors report no conflicts of interest. Authors gratefully acknowledge the financial support from CSIR Task Force Project (NWP035). R.G. thanks the University Grants Commission, New Delhi, India, for the award of a Senior Research Fellowship. ⁎Corresponding author: CSIR-Indian Institute of Toxicology Research, Lucknow-226001, U.P. India. CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, Delhi-110 007, India. E-mail addresses: [email protected], [email protected] (K.C. Gupta).
To address these limitations, Chang et al 11 modified chitosan by tethering histidine moieties onto the chitosan backbone and showed enhanced transfection efficiency due to the increase in buffering capacity in the pH range of endosomes and lysosomes. However, the cellular uptake was found to be lower than that of Lipofectamine (Invitrogen Inc., Carlsbad, California), a commercially available transfection reagent. Jiang et al 12 prepared chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer via condensation of periodate-oxidized chitosan and bPEI(1.8kD) followed by reduction of the resulting Schiff's bases. They demonstrated comparable transfection efficiency of the grafted polymer with that observed with Lipofectamine. A number of other modifications, such as trimethylation, 13 PEGylation, 14,15 depolymerization 16,17 and galactosylation, 18 have also improved the transfection efficiency of chitosan with limited success. Alternatively, in an elegant fashion, Chen et al 19,20 have employed a dissimilar approach in exploring the transport mechanism of PEG-modified polylysine compacted DNA NPs using lipid raft-mediated endocytosis to enter the cells. We, therefore, proposed to better the transfection efficiency of chitosan well above the reported success by introducing a
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.06.022 Please cite this article as: S.K. Tripathi, R. Goyal, P. Kumar, K.C. Gupta, Linear polyethylenimine-graft-chitosan copolymers as efficient DNA/siRNA delivery vectors in vitro and in vivo. Nanomedicine: NBM 2012;8:337-345, doi:10.1016/j.nano.2011.06.022
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cationic polymer onto the chitosan backbone using a novel approach. The projected grafting circumvents all the limitations (solubility, buffering and DNA binding properties) and simultaneously maintains the integrity of the chitosan backbone. For this purpose, we deliberately selected a low molecular weight lPEI(2.5kD), which possesses an adequate amount of buffering capacity but is a poor transfection reagent. 21,22 In this study, we prepared chitosan- lPEI(2.5kD) (CP) copolymers by partially converting chitosan hydrochloride into its chlorohydrin, which further partially reacts with 2° amines of lPEI(2.5kD) to convert them into 3° by keeping the total number of amines intact in the resulting copolymer. By doing so, the lPEI(2.5kD) grafted copolymer then had all types of amines (1°, 2°, 3°) that are required for good buffering, proper binding and release of DNA. 23-25 Adopting this strategy, a small series of graft copolymers (CP-1 to CP-5) was prepared by varying the amount of lPEI(2.5kD). The resulting CP copolymers, soluble at physiological pH, were subsequently characterized spectroscopically and evaluated in terms of their buffering capacity, DNA binding and release capability, the ability to carry nucleic acids in vitro and in vivo, and cytotoxicity. 26 One of the graft copolymers among the series, CP-4, demonstrated ~ 2 – 24-fold higher gene delivery ability in comparison with native chitosan, lPEI(2.5kD), bPEI(25kD) and selected commercial transfection reagents, such as Superfect (Qiagen SAS, Courtaboeuf, France) and Lipofectamine.
Methods Preparation of chitosan chlorohydrin (CC) Chitosan (100 mg, 1 mg/ml in 1% aqueous HCl) was dissolved by keeping it overnight on stirring. The resulting chitosan hydrochloride solution (100 ml) was stirred at 65°C and to it added epichlorohydrin (0.25ml). Stirring was continued for ~ 20 h at the same temperature and then concentrated to dryness on a rotary evaporator to obtained chitosan chlorohydrin hydrochloride (CC) in ~ 95% yield as a white powder. Preparation of CP CC (25 mg) and lPEI(2.5kD) (25 mg) were suspended in water (25ml, 1mg/ml) and heated to 65°C. An aqueous solution of sodium hydroxide (2 N, 2 ml) was added dropwise to the above rapidly stirred reaction mixture. The reaction was allowed to stir for ~ 20h at 65°C. Thereafter, the resulting solution was concentrated on a rotary evaporator and dialyzed against water for two days with intermittent change of water. The dialyzed solution was lyophilized in a speed vac to obtain CP-1 copolymer in ~ 85% yield. Similarly, other copolymers with increasing amounts (weight ratio) of lPEI(2.5kD), viz., 1:2 (CP-2), 1:3 (CP3), 1:4 (CP-4) and 1:5 (CP-5) were prepared in ~ 78 – 85% yield and characterized by 1H-NMR (DRX-300, 300 MHz, FTNMR, Bruker Avance, Madison, Wisconsin). Buffering capacity Chitosan, bPEI(25kD), lPEI(2.5kD) and CP copolymers (3 mg each, separately) were dissolved in 0.1 N NaCl to obtain
concentrations of 0.1 mg/ml. Aqueous HCl (0.1 M) and NaOH (0.1 M) were used as titrators. The buffering capacity of each of the polycations was determined using pH titration from low to high. Endosomal escape through confocal microscopy Endosomal escape of CP-4 and chitosan was monitored on HeLa cells. Endosomes were labeled with Alexa Fluor 488 (100 μg/ml) and lysosomes with Lysosensor Green DND-189 (1 μM) and Lysotracker Green DND-26 (1 μM). These antibodies were added to the cells prior to addition of the chitosan and CP-4 complexes containing tetramethylrhodamine-labeled pDNA, and incubated for 20 minutes in OptiMEM I medium. Subsequent to the addition of the complexes, the cells were incubated for 1h and then washed with 1 × phosphate buffered saline (PBS) (2 × 1 ml) followed by fixing with 4% paraformaldehyde. The images were recorded using a ZEISS LSM 510 inverted microscope. In vitro transfection GFP plasmid transfection To evaluate the gene-carrying efficiency of CP copolymers, HEK293, CHO and HeLa cells were seeded 1 day prior to transfection assay in 96-well plates after which media was aspirated and cells were washed with PBS (1 × PBS). CP copolymers/bPEI(25kD)/lPEI(2.5kD)/chitosan were complexed with pDNA (0.3 μg) at w:w ratios of 1:1, 1.66:1, 2.33:1, 3.3:1, 4:1 and 5:1. Similarly, pDNA complexes were prepared with the standard transfection reagents, viz., Superfect, Fugene (Roche Inc., Branchburg, New Jersey), GenePORTER 2 (Genlantis Inc., San Diego, California) and Lipofectamine, following manufacturers' protocols. DNA complexes were diluted with serum-free DMEM (60 μl) and gently added to HEK293, CHO and HeLa cells. In another set, DNA complexes were diluted in DMEM supplemented with 10% FBS and added to various cells. After 4 h, the transfection medium was replaced by 200 μl fresh DMEM containing 10% FBS and cells were further incubated for 36 h. EGFP expression in mammalian cells was quantitatively estimated on NanoDrop ND- 3000 spectrofluorometer (Thermo Scientific, Wilmington, Delaware) after 36 hours. Similarly, for FACS analysis, HEK cells were seeded in 24well plates 1 day prior to transfection and the analysis was performed 36 h post transfection. CLSM Intracellular trafficking studies of CP-4/DNA and chitosan/ DNA complexes were performed on HeLa cells. For this, pDNA was labeled with YOYO-1 iodide; CP-4 and chitosan were labeled with tetramethylrhodamine isothiocyanate (TRITC). Briefly, YOYO-1 iodide (2 μl, 1 mM solution in DMSO) was added to pDNA (0.3 mg) and the resulting mixture was kept stirring for 2h at 25 ± 1°C in dark conditions and finally stored at -20°C. CP-4 and chitosan (10 mg/ml) were treated overnight with TRITC (26 μl, 1 mg/100 μl DMF) for 1% blockage of total amines in polymers. Subsequently, the reaction mass was concentrated in a speed vac and unreacted/hydrolyzed TRITC was removed by trituration with ethyl acetate (3 × 2ml). The resulting product was completely dried and resuspended in water.
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Table 1 Particle size and zeta potential measurements of CP/DNA nanoplexes in water and serum at w/w ratio of 2.33:1 and lPEI(2.5kD)/DNA and chitosan/DNA complexes at 1.66:1. PDI = 0.15 – 0.34 S. No.
1. 2. 3. 4. 5. 6. 7.
Sample ID
CP-1 CP-2 CP-3 CP-4 CP-5 Chitosan lPEI (2.5kD)
Average particle size in nm ± SD
Zeta potential in mV ± SD
DNA-loaded copolymer (in H2O)
DNA-loaded copolymer (in DMEM)
Copolymer (in H2O)
DNA-loaded copolymer (in H2O)
DNA-loaded copolymer (in DMEM)
100 ± 1.52 90.4 ± 1.56 85 ± 3.68 75.6 ± 3.26 65.5 ± 3.52 136.3 ± 5.38 128.7 ± 4.21
85 ± 7.2 75 ± 6.4 68 ± 1.5 60 ± 3.7 55 ± 4.4 60 ± 4.3 61 ± 7.0
44.7 ± 0.87 48.3 ± 4.82 50.3 ± 0.96 53.2 ± 2.96 56 ± 1.73 30.5 ± 2.0 25.7 ± 1.17
39.7 ± 1.3 42.3 ± 1.8 45.7 ± 1.3 44.1 ± 1.6 50.8 ± 1.4 26.8 ± 2.1 22.1 ± 1.4
-11.56 ± 2.2 -10.23 ± 1.6 -9.38 ± 1.3 -11.86 ± 1.58 -10.76 ± 1.69 -9.48 ± 1.48 -9.78 ± 1.36
HeLa cells were seeded (1.5 × 10 5 cells/well) on circular glass coverslips in 6-well plates, grown overnight to ~ 70% confluence. A polyplex solution (500 μl) containing DNA (0.3 μg) in DMEM was added to each well. After incubation for 30, 60 and 120 minutes, the cells were washed with 1 × PBS (3 × 1 ml) and fixed with 4% paraformaldehyde solution for 10 minutes at 37°C. The fixed cells were counter-stained with a blue nuclear dye, DAPI, and the coverslips were mounted on glass slides with fluorescence-free Mounting Medium (UltraCruz, Santa Cruz Biotechnology, Santa Cruz, California). All confocal images were acquired using a Zeiss LSM 510 inverted confocal laser-scanning microscope (CLSM). Endosomal and lysosomal escape of complexes was also monitored by confocal microscopy. The detailed experimental procedure is given in the Supplementary Information found in the online version of this article.
Weight ratio of copolymer/DNA complexes
2.33:1 2.33:1 2.33:1 2.33:1 2.33:1 1.66:1 1.66:1
Results
chlorohydrine and CP copolymers were synthesized, as described above and depicted in Scheme-S1 (see Supplementary Information), in ~ 95 and ~ 78 – 85% yield, respectively. The degree of grafting of chlorohydrin followed by lPEI(2.5kD) on synthesized copolymers was determined by 1H-NMR spectroscopy. From integration of signals, the substitution of 3-chloro-2-hydroxypropyl groups onto a chitosan backbone was found to be 45%, i.e., ~ every two units of glucosamine contain one 3-chloro-2-hydroxypropyl group. Further, the percent substitution of linear PEI onto chitosan chlorohydrin is shown in Table S1 (see Supporting Information). The size and surface charge of the particles formed with DNA was measured by DLS and zeta sizer. The size of CP-1 to CP-5 and their pDNA complexes was measured in water and 10% serum at a weight ratio of 2.33:1 (copolymer:DNA) for the CP series, 1.66:1 for chitosan and lPEI(2.5kD). In water, moving down the series from CP-1 to CP-5, size of the particles was found to be inversely proportional with the concentration of lPEI(2.5kD) (data not shown). The same trend was observed on complexation with pDNA, though a further decrease in size of the nanoplexes may be due to the formation of more compact complexes (Table 1). In presence of serum, an additional decrease in the size of the particles was observed from CP-1 to CP-5. Unlike size, the zeta potential of the particles increased upon increasing the amount of lPEI(2.5kD) (ranging from ~ +44.7 - +56mV) in the copolymers. However, the zeta potential of particles decreased after complexation with pDNA and it became negative in presence of serum. To establish the pDNA binding ability of the copolymers, a gel retardation assay was carried out at different weight ratios. The binding of CP copolymers to DNA neutralizes the negative charge in the phosphate backbone, which resulted in the decreased mobility of DNA under the influence of electrical current. We observed that on increasing the amount of lPEI (2.5kD) in CP copolymers (CP-1 to CP-5), the electrophoretic mobility of DNA decreased with lesser amounts of copolymers and at a copolymer:DNA ratio of 1:1 (w:w) in the case of CP-4 and 5, the mobility of pDNA was completely retarded (Figure S1, Supporting Information). lPEI(2.5kD) also retarded the DNA mobility at the same weight ratio.
Preparation and characterization of CP copolymers
Buffering capacity and endosomal release
Chitosan-lPEI(2.5kD) copolymers (CP-1 to CP-5) were prepared by increasing the amount of lPEI(2.5kD). Chitosan
The endosomal release of DNA nanoplexes in the cytoplasm is one of the important parameters that depends on the intrinsic
In vivo gene expression in Balb/c mice pGL3 (25μg) was complexed with CP-4 at a w:w ratio of 1:2.33 and bPEI(25kD) at 1:1.66, and the final volume was made up to 100 μl using normal saline. The complexes were incubated for 30 min at 25 ± 1°C and injected into 3 Balb/c mice through the tail vein using an insulin syringe (1 ml) with needle size of 0.30 × 8mm. The animals were kept for 7 days on a normal diet, after which they were sacrificed by cervical dislocation and all the vital organs were dissected out. The organs were washed with chilled normal saline, weighed, chopped and made 25% w/v homogenate in 1 × cell lysis buffer (Promega Corp., Madison, Wisconsin) containing 1 × protease inhibitor cocktail. After 3 cycles of freezing and thawing in liquid nitrogen and at 37°C, respectively, the whole homogenate was centrifuged at 10,000 rpm for 10 minutes at 4°C. Cell lysate (100 μl) from each sample was assayed for luciferase activity by adding 100 μl luciferin substrate on the luminometer (Berthold Technologies, Pforzheim, Germany). The standard curve was drawn using luciferase enzyme provided with the kit. The same experiment was repeated twice.
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Figure 1. (A) Buffering capacity of lPEI(2.5kD), chitosan and CP copolymers. (B) Percent transfection efficiency in the presence of Bafilomycin A1 (Proton pump inhibitor) of bPEI(25kD), chitosan and CP-4 copolymer.
buffering capacity of the vector system, which is measured by acid-base titration. The standard acid-base titration of lPEI (2.5kD) showed a considerable buffering capacity over a pH range of 3 to 10; however, due to limited solubility of CP copolymers at higher pH, the titration was carried out over a pH range of 3 to 7.5. The volume of NaOH required for bringing the pH from 3 to 7.5 in the case of lPEI(2.5kD) and bPEI(25kD) was significantly higher (P b 0.05) than the volume required for chitosan (Figure 1, A). It is interesting to note that for CP copolymers, the volume of NaOH required was comparable with that of lPEI(2.5kD) and bPEI(25kD). Effect of H +-ATPase inhibitor The effect of Bafilomycin A1 (a proton pump inhibitor) was observed on the transfection efficiency of CP-4/DNA, bPEI (25kD)/DNA and chitosan/DNA on HEK293 cells using FACS. A considerable decrease in transfection efficiency was noticed in the case of CP-4 (~ 47% ca. ~ 8% with Bafilomycin A1) and bPEI(25kD) (~ 25% ca. ~ 6% with Bafilomycin A1), whereas the decrease in the case of chitosan (~ 2% ca. ~ 1.6% with Bafilomycin A1) was insignificant (Figure 1, B). Toxicity assessment The cytotoxicity of CP copolymers /DNA complexes at different weight ratios was investigated using MTT assay on HEK293, HeLa and CHO cells and compared with lPEI(2.5kD), bPEI(25kD), chitosan and the commercial transfection reagents. In that cytotoxicity investigation, bPEI(25kD) showed significant cytotoxicity (P b 0.05) in comparison with lPEI(2.5kD), chitosan and CP copolymers. However, lPEI(2.5kD) showed minimal cytotoxicity at its best working concentration on all the cell lines (Figure 2). Chitosan and CP copolymers did not show any cytotoxicity as evidenced by the healthy growth of the cells with more than 100% cell viability. Transfection efficiency measurements of CP copolymers Using a plasmid containing enhanced green fluorescent protein (EGFP) as the reporter gene, we compared the
Figure 2. Cell viability profile of CP/DNA nanoplexes at an w/w ratio of 1:2.33, lPEI(2.5kD)/DNA and chitosan/DNA polyplex at w/w ratio of 1:1.66, and Superfect/DNA, Fugene/DNA, GenePORTER 2/DNA and Lipofectamine/DNA complexes in HEK293, HeLa and CHO cells. Cells were treated with DNA complexes under conditions described under transfection assay, and cytotoxicity was determined by MTT assay. Percent viability of cells is expressed relative to control cells. Each point represents the mean of 3 independent experiments performed in triplicate. **P b 0.01 vs. PEI and Lipofectamine in all the cell lines.
transfection efficiency of CP copolymers with those of lPEI (2.5kD), bPEI(25kD), chitosan and selected commercial transfection reagents, viz., Superfect, Fugene, GenePORTER 2 and Lipofectamine. The same w:w ratio of copolymer:DNA (see gel retardation assay) was used in all the transfection experiments. Thirty-six hours post transfection, EGFP fluorescence was observed. Following spectrofluorometric quantification, transfection efficiency of CP copolymers at w:w ratio 2.33 was found to be significantly higher (P b 0.05) than that of lPEI(2.5kD), bPEI(25kD) and chitosan at w:w ratio of 1.66 (Figure S2, Supporting Information) in the presence and absence of serum. It is interesting to note that CP-4 among the series of synthesized copolymers displayed ~ 20- and 24-fold (P b 0.05) GFP expression in HEK293 cells in comparison with chitosan and lPEI(2.5kD), respectively, in both the presence or absence of serum. Further, with CP-4, EGFP expression was ~ 1.3- to 3.0fold higher (P b 0.05) in this particular cell line in comparison with bPEI(25kD), GenePORTER 2, Superfect, Fugene and Lipofectamine in both the presence or absence of serum (Figure S2, Supporting Information). A similar trend was observed in the other cells, viz., HeLa and CHO. To find out the cell population expressing GFP, FACS analysis was performed with the cells transfected with copolymer:DNA over a range of w:w ratios. We observed a maximum percentage (~ 45%) of GFP positive cells transfected with CP-4 at a w:w ratio of 1:2.33; however, GFP expression regressed with a deviation from the above ratio (Figure 3). In contrast, chitosan and lPEI(2.5kD) showed only ~ 3 and ~ 2% GFP positive cells, respectively, at a w:w ratio of 1:1.66. Although only 26% GFP positive cells were scored after transfection with bPEI(25kD), a gold standard for the transfection studies, at a w:w ratio of 1:1.66; 16% and 35% GFP positive cells were observed following transfection with selected commercial reagents, viz., Lipofectamine and Superfect, respectively.
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Figure 3. Percent transfection efficiencies of CP-3, CP-4, and CP-5/DNA nanoplexes determined using FACS at various w/w ratios and compared with lPEI(2.5kD), chitosan, Superfect and Lipofectamine. **P b 0.01 vs. PEI (at all w:w ratios), Superfect and Lipofectamine.
siRNA Delivery efficiency The delivery of two types of siRNAs, viz., GFP specific and JNKII, was tested on HEK293 cells. The transfection was performed in a sequential manner using GFP/CP-4 system, as described above. The observed knockdown of GFP expression by CP-4/siRNA system was found to be ~ 77%, whereas only 49% knockdown was recorded with Fugene/siRNA system using nanodrop spectrofluorometry (Figure 4, A). As a proof of principle, we tested another siRNA, JNKII, which is activated by a variety of environmental stresses. 30 Using western hybridization, we observed nearly 60% knockdown of JNKII using CP-4 as a transfection agent after 36 hours of treatment, which was comparable with that observed with the commercial transfection reagent, Lipofectamine (~ 55%) (Figure 4, B). This further boosts the potential of CP-4 as a versatile gene-delivery agent. pDNA binding affinity The binding ability of CP-4, chitosan and lPEI(2.5kD) with pDNA was studied by incubating the polyplex solution with increasing amounts of heparin, a competitive anionic moiety, and the subsequent release pattern of pDNA was recorded on gel electrophoresis. As expected, only ~ 30% of bound DNA was found to be released from chitosan, even after addition of 35 U of heparin, whereas ~ 96% DNA was released from lPEI(2.5kD) in the presence of 2.5 U heparin and ~ 72% DNA was released from bPEI(25kD) with 5 U heparin. It is interesting to note that we observed ~ 92% of DNA released from CP-4 copolymer in the presence of 5 U of heparin (Figure S3, Supporting Information).
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Figure 4. (A) CP-4 copolymer was checked for its ability to deliver siRNA in HEK293 cells by transfecting pEGFP DNA followed by GFP specific siRNA. The expression of GFP in cells was suppressed by ~ 77%, in comparison with Fugene/siRNA, which suppressed GFP expression by only ~ 49%, as monitored by measuring fluorescence on a spectrofluorometer. All the experiments were performed at least thrice and error bars represent the SD, (B) Immunoblotting assay for CP-4 to see the JNKII knockdown. Lane 1: CP-4/JNKII siRNA, lane 2: CP-4 without siRNA, Lane 3: Lipofectamine without siRNA, Lane 4: Lipofectamine/JNKII siRNA.
complexed with CP-4 copolymer did not show any significant degradation (~ 15% after 2h). Intracellular trafficking To investigate the path of cellular entry, tetramethylrhodamine-labeled CP-4 was complexed with YOYO-1-labeled pDNA and added on to HeLa cells, which were fixed with paraformaldehyde at specified time intervals. We observed that CP-4 was able to carry pDNA to the insides of the cells (cytoplasm) within 30 minutes of the addition to the cells. Further, both pDNA and CP-4 were observed inside the nucleus after 1h of the addition of the complexes, and after 2 h, the maximum amount of fluorescence was observed inside the nucleus only (Figure 5). HeLa cells, prelabeled with endosomal and lysosomal dyes (green), were incubated with tetramethylrhodamine (TMR) labeled pDNA/chitosan and CP-4 complexes (red). After imaging through confocal microscopy, it was observed that in case of chitosan, red and green regions were overlapping, showing the entrapment of these complexes within these compartments (Figure S5, Supporting Information), whereas, in the case of CP-4, separate red and green regions were observed, indicating the release of these complexes from these compartments.
Protection of pDNA against nucleases Having evaluated the binding and release of DNA from the copolymer, CP-4, it becomes mandatory to examine the integrity of pDNA bound to copolymer against nucleases. DNase I protection assay was performed and the results analyzed by 0.8% agarose gel electrophoresis. Figure S4 (Supporting Information) clearly depicts that whereas naked DNA (0.3 μg) was found to be degraded within 15 minutes of incubation with DNase I, pDNA
Protein adsorption assay The amount of protein adsorbed onto the surface of CP-4 was evaluated on SDS-PAGE taking BSA as a standard. We observed significantly reduced binding of BSA with our test copolymer, CP-4, as evidenced in Figure S6 (Supporting Information), indicating a reduction in non-specific binding of CP-4 to serum proteins.
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Figure 5. Confocal microscopic images of HeLa cells treated with tetramethylrhodamine-CP-4/YOYO-1-pDNA nanoplex at different time points.
In vivo gene expression in Balb/c mice Having achieved the significant level of transfection in vitro, we further examined the efficacy of CP-4 for its in vivo applications and used a firefly luciferase expression-reporter system in Balb/c mice. We observed a maximum luciferase activity in spleen (P b 0.05) followed by heart and brain in the mice after 7 days in comparison with bPEI(25kD) (Figure 6). We did not use chitosan and lPEI(2.5kD) in this study because none of those showed any significant transfection in vitro. Discussion Figure 6. In vivo gene expression analysis in Balb/c mice 7 days post i.v. injection using pGL3 control vector as a reporter gene. The mice were sacrificed and various organs such as heart, liver, spleen, kidney, brain and lungs were dissected out. The organs were homogenized in lysis buffer and the lysate was read on luminometer to quantify the luciferase activity. *P b 0.05 vs. PEI in the respective organs.
In this study, we tried to improve the transfection efficiency of chitosan and while designing the strategy for the preparation of modified chitosan, we were guided by four important considerations, viz., (i) the modified chitosan should be soluble at physiological pH, (ii) it should have improved buffering capacity so that desired therapeutics is released in cytoplasm, (iii) it should efficiently release DNA in the cytoplasm for nuclear
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localization, and (iv) the modified analogue of chitosan should retain the property of chitosan with respect to biocompatibility and cell viability. PEIs are known to possess very good buffering capacity; however, they exhibit molecular weight-dependent transfection efficiency. Branched PEIs with high molecular weight have better gene transfer capability but suffer from chargeassociated toxicity. PEIs with low molecular weight are nontoxic but offer poor transfection efficiency. To take advantage of the stated properties of low molecular weight PEIs and to satisfy all the above criteria, we deliberately selected low molecular weight cationic polymers (bPEI1.8kD and lPEI2.5kD) for grafting on chitosan using a two-step methodology. In the first step, the chitosan hydrochloride was converted into its chlorohydrin derivative by the reaction with epichlorohydrin, which was subsequently reacted with 1° and 2° amines of bPEI(1.8kD) via in situ generation of epoxide under alkaline conditions followed by the opening of the epoxide ring to prepare a series of bPEI(1.8kD)-graft-chitosan copolymers by varying the amount of bPEI(1.8kD). The resulting copolymers were tested for their in vitro transfection efficiency and found to be incomparable with bPEI(25kD) (a gold standard) (data not shown). Subsequently, we prepared another series of graft-copolymers by replacing bPEI(1.8kD) with lPEI(2.5kD) in an analogous manner and made a series of graft-copolymers by varying the amount of lPEI(2.5kD) (Scheme S1, Supporting Information). The percentage of grafting was determined by 1H-NMR, which confirmed that on increasing the concentration of lPEI(2.5kD), down the series, there is a gradual increase in the substitution of lPEI (2.5kD) on the chitosan backbone (Table S1, Supporting Information). All the copolymers in the series were evaluated in terms of cell viability and transfection efficiency in a variety of cell lines. The transfection efficiency of this series was found to be dramatically improved, i.e., 2- to 24-fold higher than those of chitosan, lPEI(2.5kD), bPEI(25kD) and Lipofectamine. We, therefore, proceeded with linear chitosan-lPEI (2.5kD) copolymers for subsequent studies and evaluated them for buffering capacity, DNA release and in vitro and in vivo gene expression. The size and surface charge of NPs are the vital parameters to determine their cellular uptake and interactability with the membrane. In the proposed strategy, the copolymer resulting from the mixing of two polymers should exhibit a decrease in size (due to increasing cross-linking leading to compactness) and an increase in zeta potential (due to incorporation of the more positive charge of PEI) on increasing the percent cross-linking of PEI in proceeding down the series from CP-1 to CP-5. As expected, the size of DNA complexes of CP copolymers in the presence of water and serum was found to decrease on descending the series. Such a decrease in the size of the complex in water might be due to the greater compactness of complexes on increasing the amount of PEI in the series. In presence of serum, the further decrease might be due to the adsorption of water from the complexes by the negatively charged serum proteins. Additionally, the increase in zeta potential on descending the series further substantiated the attachment of lPEI(2.5kD) with chitosan.
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The high transfection efficacy of PEI polyplexes is attributed to their excellent buffering capacity in the endosomes. 27 Conversely, due to the poor buffering capacity of chitosan, their polyplexes are unable to rupture the endosomes and are degraded into lysosomes, resulting in poor transfection efficiency. 21 On the other hand, CP copolymers were found to possess improved buffering capacities (comparable with that of PEIs) and these observations were indicative of improved transfection efficiency of CP copolymers over lPEI(2.5kD), bPEI(25kD) and chitosan. Further, to confirm the above observation, transfection assay was carried out in the presence of Bafilomycin A1 (a proton pump inhibitor), which resulted in the substantial decrease in the endosomal mediated transfection efficiency of PEI (~76%). 26 Similar results were obtained with CP-4 formulation (~ 83% decrease in the transfection efficiency); however, insignificant change in transfection efficiency was observed with the chitosan formulation. The results indicated that CP-4, having comparable buffering capacity to branched PEI(25kD), followed pathways identical to PEI, whereas chitosan, which does not possess any buffering capacity, could not rupture the endosomes. The buffering capacity was also evaluated through confocal microscopy for the CP-4 and chitosan complexes. 11 The merged green and red regions (Figure S5, Supporting Information), in the case of chitosan, showed that these complexes could not escape the endosomes and lysosomes, whereas, in the case of CP-4/ DNA complex, independent scattered red and green areas showed that these particles came out of endosomes efficiently supporting the above experiment. Prior to transfection assays, it is pertinent to test the cytotoxicity of the projected copolymers. The CP copolymers were found to possess almost negligible toxicity in comparison with bPEI(25kD) in all the cell lines tested. Because both the polymers used in this study are by themselves nontoxic to the cells, the resulting copolymers also exhibited a similar noncytotoxic effect. In addition, bPEI(25kD) was found to be toxic to the cells as it is also reported that the primary amino groups present in bPEI(25kD) cause cytotoxicity through perturbation of protein kinase activity. 28 Having confirmed the non-cytotoxic nature of the CP copolymers, we evaluated the gene transfer ability of these copolymers on a variety of cells, viz., HEK293, CHO and HeLa cells. The enhanced transfection efficiency of CP copolymers might be due to increased solubility (~ 10 mg/ml, data not shown) at physiological pH and improved buffering capacity due to lPEI (2.5kD) grafts. We also did not observe any significant change (P N .05) in transfection efficiency of CP-4 copolymers in the presence or absence of serum. This finding was further supported by an observation wherein little or negligible quantity of BSA was adsorbed onto the surface of CP-4 in comparison with lPEI (2.5kD) and chitosan (see protein adsorption assay). Therefore, diminishing interaction between serum and CP-4 could be due to the presence of hydrophilic chitosan, a sugar moiety, known for its properties to reduce interactions with serum proteins. 29 We further observed that transfection efficiency increased with the increase in the weight ratio of lPEI(2.5kD), reached up to a maximum for CP-4 and thereafter started decreasing, indicating
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CP-4 as the best carrier among the series of synthesized copolymers. Thus, in this study, the w:w ratio was found to have an important impact on the transfection ability of the synthesized copolymers and CP-4, among the tested transfection agents, emerged as the best formulation. We also established the versatility of the test copolymer, CP-4, as an efficient carrier for nucleic acids using two types of siRNAs, viz., GFP specific and JNKII on HEK 293 cells. CP-4 was able to deliver both, as could be seen by the suppression in the expression of targeted genes. To achieve successful transfection, it is a prerequisite that the bound pDNA must be released from the polyplex. The extent of binding of DNA to the cationic polymer is one of the major factors influencing transfection efficiency. Chitosan has been reported to interact with pDNA via strong electrostatic/hydrogen bonding or via hydrophobic interactions. 11 The interactions between chitosan and pDNA are quite strong and are not affected even at very high salt/detergent concentration as well as in the presence of high concentrations of heparin. 30 The binding ability was found to be in the order Chitosan⋙bPEI(25kD)NCP4NlPEI(2.5kD). These observations suggest that binding capability of CP copolymer was neither strong nor loose, which may further account for its high transfection efficiency. Sugars are known to reduce the nonspecific interactions with blood serum proteins and increase the blood circulation time. 29 To validate the same observation, the amount of protein adsorbed on to the surface of CP-4 was evaluated on SDSPAGE taking BSA as a standard. It is interesting to note that CP-4 displayed much reduced interactions with serum proteins in comparison with PEI. Sufficient protection of bound DNA from DNase I up to 2 h and reduced binding with serum proteins (BSA) suggests that the synthesized copolymer, CP-4, has the potential to be used for in vivo administration of nucleic acids. After successful transfection was achieved by CP copolymers, it was rationalized to track the path of cellular entry of DNA through confocal microscopy. The observations clearly state that the CP copolymers are capable of efficiently carrying the desired gene inside the cells. The higher uptake and transfection efficiency of CP-4 might be due to presence of sugar moieties, which helped in reducing the interactions with serum proteins present in the blood and, hence, prolonged the circulation time in the blood. 31 Luciferase activity was observed significantly in spleen, brain and heart. Spleen and liver have irregular fenestrations and also endothelia with large meshes, which allow extravasation of molecules up to 0.1 – 1 μm. 32 It was surprising that, in our experiments, negligible luciferase activity was observed in the liver and at present, we do not have any plausible explanation for that circumstance. The encouraging results of improved gene expression in the brain may be useful for brain targeting but increased gene expression in spleen indicated higher clearance of the vector by this organ and as a result shortened gene expression in the other organs. Although several reports are available on the expression and tissue distribution of the test gene(s), 17,33,34 little is known about the mechanism of distribution. Future studies would be undertaken in our laboratory to enhance understanding of the mechanism and the explanation for it. The results
presented here may be useful for the biologists working in the same field. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.nano.2011.06.022.
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