Abstract
Background: RNA interference has promising therapeutic potential. However, safe and efficacious delivery systems are necessary for its application in the clinic. Materials and Methods: An oleic acid (OA) derivative of branched polyethylenimine (PEI, M.W. 2000 Da), PEI-OA, was synthesized and evaluated for small interfering RNA (siRNA) delivery in SK-HEP-1 liver cancer cells stably transfected with luciferase. The physiochemical properties of PEI-OA/siRNA complexes, their cellular uptake, gene silencing activity based on luciferase reporter gene down-regulation, and cytotoxicity were investigated. Results: PEI-OA complexes effectively delivered siRNA into SK-HEP-1 cells and efficiently induced down-regulation of luciferase reporter gene expression. Compared with free siRNA and PEI/siRNA, PEI-OA/siRNA was significantly more effective, reducing luciferase activity by ~50%. Conclusion: PEI-OA warrants further evaluation for therapeutic delivery of siRNA.
Small interfering RNAs (siRNAs) are composed of a duplex of RNA oligonucleotides, each typically containing 21 to 23 nucleotides (1). siRNAs can be used to silence specific target genes through incorporation into RNA-induced silencing complexes (RISCs) (2-4). siRNAs have reached preclinical validation in several disease models and some have advanced into early-stage clinical trials (5-7). However, due to their large size, high charge load, and sensitivity to ribonucleases, delivery of siRNAs remains a continual challenge. Non-viral siRNA delivery systems usually are based on cationic lipids (8, 9) or cationic polymers (10-12) that form electrostatic complexes with anionic siRNA. Among cationic polymers, polyethylenimine (PEI), over a wide range of molecular weights, has been shown to allow a relatively high level of transfection efficiency. However, because of its high density of positive charge, the use of PEI is often associated with high toxicity.
To overcome this dilemma and in order to improve transfection, numerous approaches have been investigated by chemically modifying PEI (13-15). For instance, chitosan-graft-(PEI-β-cyclodextrin) (CPC) cationic co-polymers were synthesized via reductive amination between periodate-oxidized chitosan (CTS) and low molecular weight PEI-modified β-cyclodextrin (β-CD-PEI) (16). The modified PEI conjugates showed considerably higher transfection activity, as well as reduced cytotoxicity compared to unmodified PEI.
In this study, a PEI-oleic acid conjugate, PEI-OA, was synthesized, characterized, and evaluated for siRNA delivery.
Materials and Methods
Materials. Silencer Firefly luciferase (GL2+GL3) siRNA (Luciferase siRNA) and Cy3 Labeled Negative Control No. 1 siRNA (Cy3-siRNA) were purchased from Ambion, Inc. (Austin, TX, USA). Branched PEI (M.W. 2000 Da), triethylamine, and oleoyl chloride (OC, 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dichloromethane and diethyl ether were obtained from Fisher Scientific (Fairlawn, NJ, USA). Reagents were used without further purification prior to synthesis.
Synthesis and characterization of PEI-OA. PEI-OA was prepared by N-acylation of the PEI amines by OC at an OC:PEI ratio of 1:3 (wt/wt). Briefly, 32 mg of PEI was dissolved in 2.5 ml dichloromethane, containing 200 μl of triethylamine in a nitrogen atmosphere at room temperature. Ten milligrams of OC dissolved in 2.5 ml dichloromethane were slowly added to the PEI solution over a period of 30 min. After 12 h, three volumes of diethyl ether were used to precipitate the resultant product. The product was washed three times with diethyl ether and then dried under vacuum.
Particle size and zeta potential measurement. The mean particle diameter of PEI-OA micelles was measured by dynamic light scattering on a NICOMP 370 Particle Sizer (Santa Barbara, CA, USA). Zeta potentials (ζ) of PEI-OA/siRNA complexes were measured following dilution in deionized water on a Brookhaven 90plus Particle Analyzer (Holtsville, NY, USA).
Effect of N/P ratio on particle size and zeta potential of PEI-OA/siRNA complexes.
Analysis of PEI-OA/siRNA complexes by agarose gel. PEI-OA was combined with Luciferase siRNA at varying nitrogen/phosphate (N/P) ratios. Aliquots of the samples were then combined with loading buffer and loaded onto a 2% agarose gel, with ethidium bromide staining. The gel was run on a Fisher Biotech Electrophoresis System (Fairlawn, NJ, USA) at 100mV for 30 min, and was visualized on a BioRad Gel imaging system (Segrate, Milan, Italy)
Cell culture. SK-HEP-1 human liver cancer cells stably transfected with luciferase were cultured in Dulbecco's modified eagle's medium (DMEM) (Invitrogen, Grand Island, NY, USA) containing G418 and supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/ml penicillin. The cells were cultured in an atmosphere containing 5% CO2 at 37°C.
Cellular uptake determination by flow cytometry. Cells were treated with fluorescent PEI-OA/Cy3-siRNA complexes as follows. A total of 8×104 cells per well with 400 μl of DMEM were seeded in a 24-well plate prior to treatment. After 24 h of incubation and washing with FBS-free DMEM, the cells were treated with PEI-OA/siRNA complexes synthesized at N/P ratios of 1 to 8 for 1, 2, or 4 h. After treatment, cells were washed with DMEM, harvested, fixed in 4% paraformaldehyde, and analyzed on an EPICS XL flow cytometer (Beckman Coulter Inc., CA, USA).
Laser-scanning confocal microscopy. SK-HEP-1 cells were treated with either naked or PEI-OA-complexed siRNA for 4 h at 37°C. Cellular nuclei were stained with Hoechst 33342 (Invitrogen) for 5 min at room temperature. Red fluorescence of Cy3-siRNA and blue fluorescence of Hoechst were observed under the red and the blue channels, respectively, of an Olympus FV1000 Confocal Microscope (Olympus Optical Co., Tokyo, Japan).
Effect of N/P ratio on electrophoretic mobility of PEI-OA/siRNA complexes.
Cytotoxicity study. SK-HEP-1 cells were plated at a density of 104 cells/well in 96-well microtiter plates. PEI-OA/siRNA complexes synthesized at varying N/P ratios were added. The plates were then incubated for 48 h. Twenty microliters of the aqueous non-radioactive cell proliferation assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent were then added to each well. After 1 h at 37°C, absorption at 490 nm was measured on a plate reader. Experimental results are presented as a percentage of viability with respect to that of the untreated cell samples, referenced as 100%.
Luciferase down-regulation in SK-HEP-1 cells. For luciferase gene silencing, SK-HEP-1-luciferase cells were transferred to a 96-well plate. The cells were treated with either naked siRNA or PEI-OA/siRNA complexes for 4 h at 37°C. The cells were then washed with MEM and cultured for another 24 h. Then, to each well, 20 μl lysis buffer was added. The plate was shaken for 30 min. This was followed by addition of luciferase assay reagent and light measurement on a Tecan luminescence plate reader (Boston, MA, USA).
Statistical analysis. All experiments were carried out in triplicates. Statistically significant differences were analyzed with Microsoft Excel 2007 software (Microsoft, Redmond, WA, USA). The significance levels of differences from the control value were determined with Student's t-test. When p-values were <0.05 and <0.01, results were respectively considered significantly different and very significantly different.
Results
Characterization of siRNA complexes. Particle size and zeta potential analyses were performed on complexes of PEI-OA/siRNA complexes formed at N/P ratios of 1, 2, 4, 6 and 8, respectively. As shown in Figure 1 a, the complexes displayed increasing particle sizes that were inversely proportional to the N/P ratios. However, at high N/P ratios (over 6), the size became independent of the ratio. The zeta potential values are shown in Figure 1 b.
Complex formation was analyzed by agarose gel retardation assay. Complete complexation of siRNA by PEI-OA was observed at N/P ratios above 6, as shown in Figure 2.
Uptake of PEI-OA/Cy3-siRNA complexes by SK-HEP-1 cells. Cells were incubated with siRNA alone or PEI-OA/siRNA complexes prepared at a series of N/P ratios for 1, 2, or 4 h at 37°C, the cells were then washed 3 times with PBS and then analyzed by flow cytometry on a Beckman Coulter EPICS XL.
Intracellular distribution of PEI-OA/Cy3-siRNA complexes in SK-HEP-1 cells. Cells treated with PEI-OA/Cy3-siRNA complexes (with an N/P ratio of 6) were analyzed by confocal microscopy. A: Blue channel showing nuclei stained with Hoechst. B: Red channel showing Cy3-siRNA. C: Bright field image under phase contrast. D: Merged image.
Uptake of siRNA by SK-HEP-1 cells. SK-HEP-1 cells were incubated with free Cy3-siRNA or PEI-OA/Cy3-siRNA complexes at 37°C for 1, 2, or 4 h. Cellular uptake was evaluated by flow cytometry. Mean fluorescence intensities (MFIs) of cells treated by PEI-OA/Cy3-siRNA complexes were found to be much higher than those treated by free Cy3-siRNA (p<0.05) (Figure 3).
To further study the intracellular delivery pathway of Cy3-siRNA into cells, confocal microscopy was conducted after SK-HEP-1 cells were incubated with PEI-OA/Cy3-siRNA complexes for 1 h at 37°C (Figure 4). The results showed extensive internalization of Cy-3 siRNA indicating endocytosis as the major pathway of intracellular siRNA delivery.
Cytotoxicity of free Cy3-siRNA and PEI/Cy3-siRNA complexes. Cellular viability was evaluated by the MTS assay. Error bars indicate one standard deviation (n=3).
Cytotoxicity study. Cytotoxicity of free Cy3-siRNA and PEI-OA/Cy3-siRNA complexes was evaluated by the MTS assay. In this study, none of the PEI-OA/Cy3-siRNA complexes caused significant cytotoxicity compared with untreated SK-HEP-1 cells (p>0.05) (Figure 5). However, the unmodified PEI exhibited considerable cytotoxicity (p<0.05). In summary, PEI-OA/siRNA complexes have a low level of cytotoxicity.
Down-regulation of luciferase by PEI-OA/siRNA complexes. The effects of free luciferase siRNA and luciferase siRNA delivered by PEI-OA complexes were evaluated in SK-HEP-1-luciferase cells. Free siRNA was used as a control. Figure 6 shows that the luciferase level was significantly reduced when the siRNA was delivered by PEI-OA complexes. The most efficacious down-regulation was achieved at a PEI-OA/siRNA complexes formed at an N/P ratio of 6.
Discussion
Since the discovery of RNA interference in 2001 (17), there have been efforts to develop siRNAs as therapeutic agents against HIV (18), hepatitis C (19), and cancer (20). However, due to their relatively large size, rapid deactivation, and negative charge, development of more efficient delivery system for siRNA is required.
High M.W. PEI (25,000 Da) is one of the best known transfection agents for nucleic acid delivery. However, it is cytotoxic and not very effective for siRNA delivery (21). Low molecular weight PEIs are less cytotoxic, but also less efficacious (22). In our study, we synthesized a PEI-OA conjugate and evaluated its ability to deliver siRNA into SK-HEP-1 cells in vitro. The results showed potent gene silencing by PEI-OA/siRNA complexes. PEI-OA has the characteristics of a multivalent polyamine-based cationic lipid. PEI has a larger number of tertiary amines, which can be protonated at endosomal pH. This provides an efficient mechanism of endosomal escape for siRNA following internalization by endocytosis. Since PEI-OA presumably forms micelles, this represents an increase in effective size compared to unmodified PEI and an increase in the amount of positive charge per particle relative to a single PEI molecule. This should greatly increase electrostatic interactions with siRNA and the stability of the resulting complexes. Furthermore, PEI-OA has the characteristics of a surfactant, which may facilitate the breaching of the endosomal membrane following endocytosis.
Down-regulation of luciferase by PEI-OA/siRNA complexes. SK-HEP-1-luciferase cells (stably transfected) were treated with either free siRNA, or various PEI/siRNA or PEI-OA/siRNA complexes. Error bars represent 1 standard deviation. * and ** represent levels of significant differences versus untreated control at (p<0.05) and (p<0.01) respectively.
Conclusion
A novel carrier molecule, PEI-OA, was synthesized and evaluated for delivery of siRNA in vitro. The PEI-OA/siRNA complexes exhibited high cellular uptake, efficient luciferase down-regulation, and low cytotoxicity in vitro. Further studies are warranted to characterize PEI-OA/siRNA complexes as therapeutic delivery vehicles in vivo.
Footnotes
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↵* These Authors contributed equally to this study.
- Received February 8, 2012.
- Revision received February 29, 2012.
- Accepted March 1, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
