Article pubs.acs.org/Biomac
Cell-Penetrating Magnetic Nanoparticles for Highly Efficient Delivery and Intracellular Imaging of siRNA Lifeng Qi,*,†,‡ Lixia Wu,‡ Shu Zheng,§ Yilong Wang,† Hualin Fu,∥ and Daxiang Cui*,∥ †
The Institute for Advanced Materials and Nano Biomedicine, Tongji University, 67 Chifeng Road, Shanghai, 200092, China Zhejiang California Nanosystems Institute, Zhejiang University, 268, Kaixuan Road, Hangzhou, 310029, China § The Second Affiliated Hospital, School of Medicine, College of Life Sciences, Zhejiang University, 88, Jiefang Road, Hangzhou, 310009, China ∥ Department of Bio-Nano Science and Engineering, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, National Key Laboratory of Nano/Micro Fabrication Technology, Institute of Micro-Nano Science and Technology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China ‡
ABSTRACT: RNA interference is one of the most promising technologies for cancer therapeutics, while the development of a safe and effective small interfering RNA (siRNA) delivery system is still challenging. Here, amphipol polymer and protamine peptide were employed to modify magnetic nanoparticles to form cell-penetrating magnetic nanoparticles (CPMNs). The unique CPMN could efficiently deliver the eGFP siRNA intracellularly and silence the eGFP expression in cancer cells, which was verified by fluorescent imaging of cancer cells. Compared with lipofectamine and polyethyleneimine (PEI), CPMNs showed superior silencing efficiency and biocompatibility with minimum siRNA concentration as 5 nm in serum-containing medium. CPMN was proved to be an efficient siRNA delivery system, which will have great potential in applications as a universal transmembrane carrier for intracellular gene delivery and simultaneous MRI imaging.
■
tions such as hyperthermia,15 ultrasensitive detection,16,17 drug or gene delivery,18,19 cell separation,20,21 simultaneous imaging, and targeted therapy of tumors,22−25 and real-time monitoring of drug distribution in vivo.26 Their magnetic nature allows for more efficient bioapplications by an external field gradient. Magnetic nanoparticles associated with siRNA delivery have been used to reduce retrovirus-mediated expression of luciferase in HeLa cells.27 Recently, dextran-coated magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules have been used for in vivo imaging of delivered siRNA and gene silencing in tumors.28 Mostly, polymeric coatings such as polyethylene glycol,29 polyethyleneimine (PEI),30 block copolymers,31 and so on were used to modify magnetic nanoparticles for gene or drug delivery. However, those approaches usually fabricated larger nanocomposites, which can decrease the gene delivery efficiency because of the uptake of reticuloendothelial system (RES); as a result, it can also lead to higher cytotoxicity. Cell-penetrating peptides (CPPs), for example, Tat peptide, have been used to modify cationic carriers to improve siRNA delivery efficiency.32,33 Protamine sulfate (Pro), one kind of CPP, is a low-molecular-weight (4000 Da), naturally occurring
INTRODUCTION RNA interference (RNAi) is one of the most exciting discoveries in functional genomics during the past decade. RNAi is becoming an important method for analyzing gene functions in eukaryotes, and holds great promise for the development of therapeutic gene silencing.1−3 A number of approaches have been developed for in vitro and in vivo small interfering RNA (siRNA) delivery, including liposomes,4,5 polymers,6 peptides,7 virus-based vectors, and so forth.8 However, the application of RNAi in clinical therapy is still limited due to two existing problems, rapid degradation caused by exonucleases or endonucleases, and poor diffusion efficiency across the cell membrane to reach specific tissues.9−11 Thus, developing a safe and highly efficient siRNA delivery system with enhanced siRNA stability in vivo and high specificity to the desired tissue site is still the key to solve the present problems. With the advance of nanotechnology, various nanoparticulate systems such as silica nanoparticles,12 gold nanoparticles,13 chitosan nanoparticles,14 and so on have been fabricated as siRNA delivery vehicles. Compared with other nanoparticles as siRNA delivery carriers, magnetic nanoparticles have drawn much attention due to their advantages such as simultaneous siRNA delivery with MRI imaging, enhanced cellular uptake, and potential tumor targeting by external magnetic field. Magnetic nanoparticles have been widely used in bioapplica© 2012 American Chemical Society
Received: May 3, 2012 Revised: August 11, 2012 Published: August 22, 2012 2723
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
exchanged three times to finish complete dialysis. Prepared CPMNs were sterilized by a 0.22 μM membrane and stored at 4 °C for further usage. Characterization of CPMNs. The morphology and size of the aqueous dispersion of the MNPs, CPMNs, and CPMN−siRNA complexes were characterized by using transmission electron microscopy (TEM) (JEOL JEM-2100, Japan), operating at an accelerating voltage of 200 kV. The concentration of nanoparticles for TEM and size characterization is 10 nM. The dynamic radii and ζ potential of MNP and CPMN in cell culture medium were measured by a nanoparticle size analyzer Malvern 2000 (Malvern, England). Magnetization curves of prepared magnetic nanoparticles (MNPs) were measured using superconducting quantum interference device (SQUID) magnetometer (Quantum Design, PPMS-9T). Thermogravimetric analysis (TGA) measurements were performed on a PerkinElmer Pyris 1 to determine the graft rate of protamine onto the PMAL polymer of MNP. MNP and CPMN samples were respectively analyzed at a heating rate of 10 °C/min up to 800 °C under N2. The electrophoretic mobility of the prepared nanoparticles and complexes of CPMN with different molar ratios of siRNA was determined by 0.8% agarose gel in Tris/borate/EDTA (TBE) buffer at 100 mV for 30 min. SiRNA Protection by CPMN. For siRNA stability studies, siRNACPMN complexes (10 pmol) were incubated with 25 ng Ribonuclease A (Fisher Scientific, Pittsburgh, PA) for 30 min, and the enzyme was inactivated with 1 μL of ribonuclease inhibitor (Fisher Scientific, Pittsburgh, PA). The siRNA molecules were then released from the surface of CPMN by 1% sodium dodecyl sulfate (SDS). Electrophoresis by 0.8% agarose gel in TBE buffer at 100 mV for 30 min was used to quantify the intact siRNAs. Ten nanomoles of CPMN and equal siRNA were used. In Vitro siRNA Transfection. siRNA transfection was performed with CPMN, and for comparison, with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Briefly, MCF7 and U251 cells stably expressing the eGFP gene were obtained upon lentiviral transfection, and cells at 2 × 105 /mL were plated into six-well plates overnight to achieve 60−80% confluence. For siRNA transfection with lipofectamine, 1 μL/well transfection reagent, according to the vendor’s protocol, was diluted in 50 μL of OptiMEM, incubated for 10 min at RT, and mixed with siRNA. The complexes were added into cell culture to reach final siRNA concentrations ranging from 5 nM to 33 nM. For transfection with MNP and CPMN, complete medium was used during the whole transfection process. Twenty picomoles of MNP or CPMN was diluted into 50 μL of full Dulbecco's modified Eagle medium (DMEM) medium containing 10% fetal bovine serum (FBS), then mixed with different amounts of eGFP siRNA in 50 μL and vortexed for 10 s followed by 20 min incubation at RT. When 15 nM siRNA was used for transfection by use of CPMN, the N/P ratio reached 10, which is comparable with that of lipofectamine by use of the same siRNA concentration. Scrambled siRNAs with each transfection reagent were used as the nontargeting control. The mixture of transfection reagents and siRNA (total 100 μL) was added to each well to obtain different final siRNA concentrations. To test the effects of external magnetic field on the silencing efficiency, we put one thick neodymium iron boron NdFeB disk magnet underneath the cell culture plates for 4 h. After incubation with different transfection complexes for 48 h, the cells were collected, and approximately 1 × 105 cells from each sample were subjected to flow cytometry analysis to determine the silencing efficiency against eGFP, and protein analysis was conducted by Western blot. The experiments were repeated at least three times. Cytotoxicity Evaluation. Standard MTT assay was performed to determine the cytotoxicity of the transfection agents alone and their siRNA complexes.41 Briefly, cells were incubated with complexes of transfection agents and siRNA in serum-free medium for 24 h, then collected by trypsinization, counted, and plated at a density of 2 × 104 cells/well in 96-well flat-bottomed microtiter plates (100 μL of cell suspension/well). Each siRNA delivery agent was investigated with or without siRNA. The cells were then incubated at 37 °C for 48 h, and 50 μL MTT reagent was added in each well. After another 4 h of
polycationic peptide approved by the FDA as an antidote to heparin anticoagulation. Protamine sulfate has been commonly used in gene therapeutic protocols to improve the gene transfection due to its superior biocompatibility.34 Protamine sulfate conjugated to ferumoxides have also been used for labeling and imaging of human neural stem cells while not influencing cell function.35 Magnetofection of cationic magnetic nanoparticles, in which the external application of magnetic field is used to enhance intracellular uptake of large molecules, has been used to deliver siRNA;36,37 however, the magnetofection of magnetic nanoparticles alone is relatively inefficient compared with the use of cationic lipid-based transfection agents.38 In our previous work, proton sponge polymer and amphipol were used to modify quantum dots to serve as a self-tracking carrier for siRNA delivery and real time imaging.39,40 In order to develop a safer and more efficient siRNA delivery vehicle for magnetofection, we herein prepared cell-penetrating magnetic nanoparticles (CPMNs), in conjugation with low molecular protamine peptide with magnetic nanoparticles to improve the transfection efficiency. Results showed that prepared CPMNs markedly enhanced the efficiency of siRNA delivery, and allowed intracellular siRNA release and MRI simultaneously. The system renders the possibility to serve as a universal transmembrane carrier for intracellular siRNA delivery and imaging applications.
■
MATERIALS AND METHODS
Reagents and instrument. Poly(maleic anhydride-alt-1-decene) modified with dimethylamino propylamine (PMAL) with molecular weight as 18 kDa was purchased from Anatrace, Inc. (Maumee, OH, USA). A tabletop ultracentrifuge (Beckman TL120, USA) was used for nanoparticle purification and isolation. The dry and hydrodynamic radii of nanoparticles were measured by using a CM100 transmission electron microscope (Philips EO, The Netherlands) and a nanoparticle size analyzer (NanoZS, Worcestershire, United Kingdom). Confocal fluorescence images of cells after siRNA transfection were obtained with a confocal microscope (Zeiss LSM 510, Germany) equipped with DPSS, Argon, and He/Ne lasers with lines at 405, 458, 488, 543, and 633 nm. Gel images were acquired with a macro-imaging system (Lightools Research, Encinitas, CA). Protamine sulfate and 1ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), as well as other chemical reagents, were all purchased from Sigma Aldrich. Low molecular weight protamine peptide (VSRRRRRRGGRRRR) with HPLC purity higher than 95% was synthesized by Science peptide (Shanghai, China); its molecular weight is 1880 Da. Preparation of CPMNs. Hydrophobic superparamagnetic iron oxide magnetic nanoparticles in chloroform were obtained from Nanomics Biopharm (Wuxi, China). Ten milligrams of PMAL was mixed with 1 nmol (refer to particle number) of magnetic nanoparticles in chloroform, and the solvent was then allowed to slowly dry in air, leading to the formation of a thin film of PMALmodified magnetic nanoparticles (MNPs). The dried film was dissolved in 50 mM borate buffer (pH 8.0) with agitation and sonication. Free PMAL polymers (unbound polymers) were removed by ultracentrifugation at 25 000 rpm for 30 min twice. The MNPs were conjugated with low molecular protamine peptide by using a modification of the standard EDC−NHS reaction. For details, a starting molar ratio of MNP versus protamine peptide of 1:50 was used, 1 nmol MNP was mixed with 50 nmol protamine peptide in phosphate-buffered saline (PBS), then 10 mg/mL EDC was added into the mixture of MNP and protamine for conjugation. After 2 h of conjugation at room temperature (RT), the prepared CPMNs were then purified by a dialysis membrane against 0.01 M PBS buffer with a cut off molecular weight of 6000 Da to remove unconjugated protamine peptide with a molecular weight of 1880 Da. PBS buffer was 2724
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
Scheme 1. Schematic Strategy of the CPMN Hybrid Nanoparticles for eGFP siRNA Delivery
incubation, cells were washed and 100 μL dimethyl sulfoxide (DMSO) were added to each well to dissolve unsolved MTT. The absorbance of the converted dye was measured at a wavelength of 570 nm. The experiments were repeated at least three times. The cell viability was calculated using the following formula: average A value of experimental group/average A value of control group × 100%. Morphological Observation by Confocal Microscope. Living cells were seeded at a concentration of 2 × 104 cells/chamber in 300 μL growth medium (DMEM supplemented with 10% calf serum, 1% L-glutamin, and 1% penicillin/streptomycin) in an IbiTreat μ-Slide eight-well plate (Ibidi, Munich, Germany), overnight to achieve 60− 80% confluence. Then cells were transfected with CPMN−siRNA complexes or lipofectamine−siRNA at a final siRNA concentration of 5 nM, equal with the concentration of MNP and CPMN used. After 4 h of incubation with transfection complexes, images of living cells were taken with a confocal microscope LSM 510 Meta (Zeiss, Germany). The laser lines used for excitation were 488 nm for eGFP and 550 nm for Cy3 labeled siRNA. Five nanomolar Cy3-labeled siRNA was used to examine the intracellular siRNA delivery by MNP and CPMN in MCF-7 cells without eGFP expression by use of a confocal microscope, and lipofectamine-mediated siRNA delivery was used as a positive control. Green color lysotracker (Invitrogen, USA) was used to study the colocalization of internalized siRNA and lysosomes. To remove cell surface proteoglycans, cells were incubated with 80 mM sodium chlorate (NaClO3) for 24 h prior to transfection experiments, and the sodium chlorate was kept throughout the entire transfection process to determine whether CPMN−siRNA complexes passed the cell membrane through binding with proteoglycans. Statistical Analysis. All data are expressed in this article as means ± SD. P values of less than 0.05 were taken to indicate statistical significance. All figures shown in this article were obtained from three independent experiments with similar results. Analyses were performed using Origin 8.0 and Exel software.
conjugated with the carboxylic group of MNPs to obtain CPMNs, which further combined with eGFP siRNA to improve the siRNA delivery efficiency. The schematic of the whole experimental strategy is shown in Scheme 1. The unique zwitterionic surface charge of PMAL has been proved to enhance the intracellular release of siRNA from the endosome track, and also can facilitate in vivo application, because zwitterionic charge can slow down nanoparticles’ uptake by the RES through reduced serum protein adsorption onto the nanoparticles’ surface.40 The dynamic laser scattering (DLS) analysis (Figure 1A−C) showed that the hydrodynamic particle sizes of the core magnetic nanoparticles before PMAL polymer modification, MNP, and CPMN were about 15, 23, and 30 nm respectively; the particle size of CPMN has no significant difference before and after mixing with siRNA. TEM images of MNP (Figure 1D) and CPMN before (Figure 1E) and after siRNA mixing (Figure 1F) showed that they were water-soluble, and dispersed well. The zwitterionic surface charge of MNPs gave them positive surface charge when dispersed in weak acid buffer with a pH value of about 6.8. PMAL-modified MNPs are hydrophilic, approximately spherical with average particle size about 20 nm (Figure 1D), and possess superparamagnetic properties at RT with a saturation magnetization (Ms) value of 42.0 emu·g−1 (Figure 1G). TGA experiments were performed to determine the graft rate of protamine peptide onto the PMAL polymer of MNP. The TGA profile of MNP shows a weight loss of about 4.3% over the temperature range from the beginning to 650 °C (Figure 1H,a), while that of CPMN experienced a higher weight loss of about 11.5% over the same temperature range (Figure 1H,b). The weight loss under 650 °C is attributed to the polymer and peptide coated on the surface of core magnetic nanoparticles. The result demonstrated that protamine peptide was successfully coated onto the surface of MNP. The start weight of samples of both MNP and CPMN was 5 mg, and the molecular weights of PMAL polymer and protamine peptide were 18000 and 1880 Da, respectively. On the basis of the
■
RESULTS AND DISCUSSION Synthesis and Characterization of CPMN. PMALs are linear polymers with alternating hydrophilic and hydrophobic side chains, which can make the hydrophobic magnetic nanoparticles water-soluble, and their carboxylic groups provide conjugation sites for protamine. PMAL-modified magnetic nanoparticles are denoted as MNPs. Protamine peptide was 2725
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
Figure 1. Physichemical characterization of MNP and CPMN. Particle size of core magnetic nanoparticles before PMAL modification (A), MNP (B), and CPMN (C) by DLS determination. TEM images of MNP (D), CPMN (E), and CPMN-siRNA complexes (F) in cell culture medium. Saturation magnetization curve of MNP (G). TGA profiles (H) of MNP (a) and CPMN (b).
different weight losses from TGA profiles, the graft molar rate of protamine peptide onto PMAL polymer was calculated as 15:1. Agarose gel electrophoresis analysis showed that CPMNs were more positively charged compared with MNPs (Figure 2A). The enhanced ζ potential of CPMN by protamine peptide provides higher binding capability of siRNA. The ζ potentials of MNPs and CPMNs measured by DLS were around 15.6 ± 5 mV and 30.5 ± 2 mV, respectively (Figure 2C). After being mixed with siRNA molecules at equal molar ratio, the ζ potential of CPMN−siRNA complexes was reduced to 26.4 ± 3 mV. When the molar ratio of CPMN versus siRNA decreased to 20, the ζ potential of CPMN−siRNA reduced to neutral. The results were consistent with siRNA binding experiments as shown in Figure 2B. Protection of siRNA by CPMN. The binding of siRNA to CPMN by electrostatic interaction provides the protection of siRNA against enzymatic degradation. It is well known that
siRNA molecules are susceptible to nuclease digestion, therefore enhanced resistance to nuclease degradation should increase siRNA lifetime in the cell and the subsequent interference effects on target mRNAs. As shown in Figure 3, agarose gel electrophoresis analysis shows that intact siRNA can be released from CPMN−siRNA complexes after being treated with RNase A, while siRNA alone could be degraded completely by RNase A. The results highly suggested that prepared CPMNs can protect the siRNA from RNase degradation efficiently compared to free siRNA molecules, and siRNA can be released from the CPMN−siRNA composites. Cytotoxicity. Regarding the cytotoxicity of CPMN−siRNA complexes in MCF-7 cells (Figure 4C) and U251 cells (Figure 4D), prepared CPMNs exhibited lower cytotoxicity than lipofectamine and PEI in serum-free medium when using a different amount of siRNA (P < 0.05). MCF-7 cells and U251 cells incubated with 1 uM CPMN for 48 h still had 90% cell 2726
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
one thick neodymium iron boron (NdFeB) disk magnet to provide the magnetic field for siRNA delivery. As shown in Figure 4A,B, compared with lipofectamine− siRNA, CPMN−siRNA complexes can strongly inhibit the expression of eGFP in MCF-7 cells and U251 cells. Flow cytometry analysis showed that the eGFP expression in MCF7 cells can be suppressed to 12% by CPMN compared with 38% expression by lipofectamine and 24% by MNP while using 33 nM eGFP siRNA (P < 0.01 among triplicate tests). Under the application of a locally external magnetic field, the siRNA concentration to knockdown the eGFP expression can be efficiently reduced 6.6 fold (33 to 5 nM) to obtain more than 88% eGFP silencing effects. Silencing effects of CPMN were also confirmed by confocal microscope and Western blot (Figure 5), showing that CPMN exhibited higher silencing effects against eGFP compared with lipofectamine and MNP groups, even by use of 5 nM siRNA. Especially, CPMN-siRNA transfection was conducted in serumcontaining medium, while for lipofectamine, it demands serumfree medium to obtain high transfection efficiency. The intracellular distribution of CPMN−siRNA complexes were observed under a confocal microscope. Cy3-labeled siRNA mainly distributed in cytoplasm and partially colocalized with lysotracker, which suggests that the uptake of CPMN−siRNA is through a lysosome internalization pathway (Figure 6). Potential Mechanism. Protamine, a CPP, can bind with cell surface proteoglycans to induce cellular internalization. Protamine-conjugated MNP may enter the cells through the same cellular internalization pathway. Cy3-labeled siRNA molecules were respectively mixed with lipofectamine, MNP, and CPMN, then incubated with MCF-7 cells for 30 min and observed by a confocal microscope. This time point allowed us to discriminate the effect of magnetofection from that of general endocytosis-mediated cellular uptake. Cationic lipidbased nanoparticles were reported to be internalized by endocytosis following contact with the cell membrane38 and decomplex more easily in cells.42 In order to improve the magnetofection of magnetic nanoparticles alone for siRNA delivery, we modified the magnetic nanoparticles by PMAL polymer, followed by protamine conjugation. Compared with lipofectamine and MNPs, CPMNs showed much higher intracellular siRNA delivery efficiency (Figure 7), while lipofectamine-mediated siRNA delivery before 30 min was mostly located on the cell membrane; MNP-mediated siRNA has shown more intracellular uptake than lipofectamine besides having some still on the membrane. This indicated that lipofectamine-based siRNA delivery requires a longer interaction with the cell membrane prior to endocytosis. By contrast, protamine peptide conjugation could enhance the intracellular siRNA delivery significantly, which is attributed to improved intracellular particle uptake by the cell-penetrating features of protamine. Sodium chlorate is a known inhibitor of proteoglycan synthesis, which can inhibit proteoglycan-dependent binding and uptake.43 To study whether CPMN can be internalized without cell surface proteoglycans, we compared the internalization of CPMN−siRNA (Cy3-labeled) by sodium chloratetreated MCF-7 cells and untreated cells. As shown in Figure 7C, untreated cells showed a large amount of CPMN−siRNA complexes localized in the cytoplasm, exhibiting strong fluorescent signals, while the uptake of CPMN-siRNA in the sodium chlorate-treated cells (Figure 7D) were strongly inhibited, exhibiting no fluorescent signals, which indicated
Figure 2. Determination of surface charge by electrophoresis and DLS determination. (A) Electrophoresis of MNP and CPMN by 0.8% agarose gel in TBE buffer at 100 mV for 30 min. (B) Electrophoresis of CPMN mixed with different molar ratios of siRNA. (C) ζ potentials of MNP, CPMN, and CMPN-siRNA complexes in accordance with electrophoresis.
Figure 3. Electrophoresis analysis of CPMN−siRNAs with or without RNase by 0.8% agarose gel at 100 mV for 30 min. SDS was used to release siRNA from CPMN after the nuclease treatment. Lane 1: siRNA only without RNase and without SDS. Lane 2: broadening siRNA band by SDS. Lane 3: Free siRNAs were completely digested by nuclease. Lane 4: siRNA was undetectable if SDS was not used to release siRNA from the surface of CPMN. Lanes 5 and 6: the difference of nuclease-treated or nontreated siRNA after releasing from the surface of CPMN.
viability (Figure 4E,F), showing that prepared CPMN had good biocompatibility. Conversely, MCF-7 cells and U251 cells incubated with lipofectamine only had around 70% cell viability; PEI showed the highest cytotoxicity compared with the other transfection reagents. RNAi Efficiency. To evaluate the RNAi efficiency using CPMN delivery vehicle, an experimental model of gene silencing was established by using MCF7 and U251 cells with stable eGFP transfection and siRNA targeting eGFP. CPMN was mixed with eGFP siRNA at optimal molar ratio, then the transfection complexes were incubated with MCF7 cells or U251 cells for 48 h. A confocal microscope was used to image the cells after eGFP knockdown, and flow cytometry was used to quantify the silencing efficiency by CPMN−siRNA complexes. In order to determine the effects of the gradient magnetic field on the silencing efficiency of CPMN, we used 2727
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
Figure 4. Silencing effects against eGFP siRNA and cytotoxicity of CPMN compared with other transfection reagents. Silencing effects against eGFP in MCF-7 cells (A) and U251 cells (B). Cell viability of MCF-7 cells (C) and U251 cells (D) after 48 h transfected with different concentrations of siRNA by different transfection reagents determined by the MTT method. Cytotoxicity of different concentrations of transfection reagents alone 48 h after being incubated with MCF-7 cells (E) and U251 cells (F). Lipo: lipofectamine; MNP: magnetic nanoparticles; CPMN: cell-penetrating magnetic nanoparticles; CPMN-Mag: CPMN mediated siRNA delivery under external magnetic field.
Figure 5. Silencing effects of 5 nM eGFP siRNA in MCF-7 cells by MNP, CPMN, and CPMN under external magnetic field (A) negative control by siRNA only; (B) CPMN with scrambled siRNA; (C) CPMN−eGFP siRNA under magnetic field; (D) lipofectamine with eGFP siRNA; (E) MNP with eGFP siRNA; (F) CPMN with eGFP siRNA.
According to data mentioned above, we suggest a mechanism model in which CPMN can bind with proteoglycans on the surface of cancer cells. The binding induces cellular internal-
that the cell surface proteoglycans might provide the binding sites for CPMN and thus are involved in CPMN-mediated siRNA delivery. 2728
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
■
CONCLUSION
■
AUTHOR INFORMATION
Article
A unique noninvasive siRNA delivery system, the CPMN system was successfully developed. Compared with traditional siRNA carriers such as lipofectamine and PEI, this new nanocarrier can silence eGFP expression more efficiently in serum-containing medium. Especially, the CPMNs can reduce the required siRNA concentration 6.6-fold under the applied external magnetic field. CPMNs can also offer a potential method for tumor drug delivery with benefits beyond the enhanced permeability and retention (EPR) effect. The system renders the possibility to serve as a universal transmembrane carrier for intracellular drug delivery and MRI imaging applications.
Corresponding Author
*Tel: 86-021-65988029, e-mail:
[email protected] (L.Q.); Tel: 86-021-62933291, e-mail:
[email protected] (D.C.). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by the NSFC (30900345), Programs Foundation of the Ministry of Education of China (20090101120155), the Startup Foundation of the Ministry of Education for Returned Scholars (J20101127), and the Foundation of the Zhejiang Provincial Department of Education (N20100475). This work was partially supported by the Nano 973 Project of China (2010CB933901).
Figure 6. Intracellular distribution of Cy3-labeled siRNA (red color) in U251 cells transfected by CPMN. (A) siRNA evenly distributed in cytoplasm; (B) distribution of lysotracker (green color); (C) phase contrast image control; (D) siRNA partially colocalized with lysotracker, shown by the merged picture of A, B, and C.
ization, and then CPMN−siRNA complexes were taken up by endosome; after rupture of the endosome membrane by functional ligands of CPMN, CPMN−siRNA complexes enter into cytoplasm, and siRNA can be released from the complexes and then combine with RNA strands, thus inhibiting RNA function. Because siRNAs can be fluorescence-labeled, cancer cells transfected with siRNA can be imaged by fluorescent microscopy.
■
REFERENCES
(1) Hannon, G.J . Nature 2002, 418, 244−51. (2) Li, F.; Martienssen, R.; Cande, W. Z. Nature 2011, 475, 244−8. (3) Meister, G.; Tuschl, T. Nature 2004, 431, 343−9. (4) Whitehead, K. A.; Matthews, J.; Chang, P. H.; Niroui, F.; Dorkin, J. R.; Severgnini, M.; Anderson, D. G. ACS Nano 2012, DOI: 10.1021/ nn301922x.
Figure 7. Intracellular distribution of Cy3-labeled siRNA complexes inside MCF-7 cells 30 min after being transfected with lipofectamine (A), MNP (B), CPMN without pretreatment of sodium chlorate (C), and control CPMN with (D) pretreatment of sodium chlorate. Result showed that pretreatment of sodium chlorate can inhibit cellular uptake of CPMN−siRNA complexes efficiently. 2729
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
Biomacromolecules
Article
(36) McBain, S. C.; Yiu, H. H.; Dobson, J. Int J Nanomedicine 2008, 3, 169−80. (37) Mykhaylyk, O.; Zelphati, O.; Rosenecker, J.; Plank, C. Curr. Opin. Mol. Ther. 2008, 10, 493−505. (38) Lee, S.; Shim, G.; Kim, S.; Kim, Y. B.; Kim, C. W.; Byun, Y.; Oh, Y. K. Nucleic Acid Ther. 2011, 21, 165−72. (39) Yezhelyev, M. V.; Qi, L.; O’Regan, R. M.; Nie, S.; Gao, X. J. Am. Chem. Soc. 2008, 130, 9006−12. (40) Qi, L.; Gao, X. ACS Nano 2008, 2, 1403−10. (41) Yang, H.; Chen, L.; Lei, C.; Zhang, J.; Li, D.; Zhou, Z.; Bao, C.; Hu, H.; Chen, X.; Cui, F.; Zhang, S.; Zhou, Y.; Cui, D. Appl. Phys. Lett. 2010, 97, 043702. (42) Yadava, P.; Roura, D.; Hughes, J. A. Oligonucleotides 2007, 17, 213−22. (43) Payne, C.; Jones, S.; Chen, C.; Zhuang, X. Traffic 2007, 8, 389− 01.
(5) Yang, X. Z.; Dou, S.; Wang, Y. C.; Long, H. Y.; Xiong, M. H.; Mao, C. Q.; Yao, Y. D.; Wang, J. ACS Nano 2012, 6, 4955−65. (6) Kulkarni, A.; DeFrees, K.; Hyun, S. H.; Thompson, D. H. J. Am. Chem. Soc. 2012, 134, 7596−9. (7) Coursindel, T.; Järver, P.; Gait, M. J. Nucleic Acid Ther. 2012, 22, 71−6. (8) Gupta, P. K.; Sonwane, A. A.; Singh, N. K.; Meshram, C. D.; Dahiya, S. S.; Pawar, S. S.; Gupta, S. P.; Chaturvedi, V. K.; Saini, M. Virus Res. 2012, 163, 11−8. (9) Liu, N.; Ding, H.; Vanderheyden, J. L.; Zhu, Z.; Zhang, Y. Nucl. Med. Biol. 2007, 34, 399−404. (10) Bartlett, D. W.; Davis, M. E. Nucleic Acids Res. 2006, 34, 322− 33. (11) Pan, B.; Cui, D.; Xu, P.; Ozkan, C.; Feng, G.; Ozkan, M.; Huang, T.; Chu, B.; Li, Q.; He, R.; Hu, G. Nanotechnology 2009, 20, 125101. (12) Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J. I.; Nel, A. E. ACS Nano 2010, 4, 4539−50. (13) Han, L.; Zhao, J.; Zhang, X.; Cao, W.; Hu, X.; Zou, G.; Duan, X.; Liang, X. J. ACS Nano 2012, DOI: 10.1021/nn3024688. (14) Chen, M.; Gao, S.; Dong, M.; Song, J.; Yang, C.; Howard, K. A.; Kjems, J.; Besenbacher, F. ACS Nano 2012, 6, 4835−44. (15) Cui, D.; Han, Y.; Li, Z.; Song, H.; Wang, K.; He, R. Nano Biomed. Eng. 2009, 1, 61−74. (16) Chen, L.; Bao, C.; Yang, H.; Li, D.; Lei, C.; Wang, T.; Hu, H. Y.; He, M.; Zhou, Y.; Cui, D. X. Biosens. Bioelectron. 2011, 26, 3246−53. (17) Hu, H.; Yang, H.; Li, D.; Wang, K.; Ruan, J.; Zhang, X.; Chen, J.; Bao, C.; Ji, J.; Shi, D.; Cui, D. Analyst 2011, 136, 679−83. (18) Pan, B.; Cui, D.; Sheng, Y.; Ozkan, C.; Gao, F.; He, R.; Li, Q.; Xu, P.; Huang, T. Cancer Res. 2007, 67, 8156−63. (19) Kamau, S.; Hassa, P.; Steitz, B.; Petri-Fink, A.; Hofmann, H.; Hofmann-Amtenbrink, M.; Rechenberg, B; von; Hottiger, M. O. Nucleic Acids Res. 2006, 34, e40. (20) Xu, H.; Aguilar, Z.; Yang, L.; Kuang, M.; Duan, H.; Xiong, Y.; Wei, H.; Wang, A. Biomaterials 2011, 32, 9758−65. (21) Perez, J. M. Nat. Nanotechnol. 2007, 2, 535−36. (22) Huang, P.; Li, Z.; Lin, J.; Yang, D.; Gao, G.; Xu, C.; Bao, L.; Zhang, C.; Wang, K.; Song, H.; Hu, H.; Cui, D. Biomaterials 2011, 32, 3447−58. (23) Perez, J. M.; Josephson, L.; O’Loughlin, T.; Högemann, D.; Weissleder, R. Nat. Biotechnol. 2002, 20, 816−20. (24) Perez, J. M.; Simeone, F. J.; Saeki, Y.; Josephson, L.; Weissleder, R. J. Am. Chem. Soc. 2003, 125, 10192−93. (25) ornara, A.; Johansson, P.; Petersson, K.; Gustafsson, S.; Qin, J.; Olsson, E.; Ilver, D.; Krozer, A.; Muhammed, M.; Johansson, C. Nano Lett. 2008, 8, 3423−28. (26) Medarova, Z.; Pham, W.; Kim, Y.; Dai, G.; Moore, A. Int. J. Cancer 2006, 118, 2796−02. (27) Schillinger, U.; Brill, T.; Rudolph, C.; Huth, S.; Gersting, S.; Krötz, F.; Hirschbergerd, J.; Bergemanne, C.; Planka, C. J. Magn. Magn. Mater. 2005, 293, 501−08. (28) Medarova, Z.; Pham, W.; Farrar, C.; Petkova, V.; Moore, A. Nat. Med. 2007, 13, 372−77. (29) Endres, T. K.; Beck-Broichsitter, M.; Samsonova, O.; Renette, T.; Kissel, T. Biomaterials 2011, 32, 7721−31. (30) Bain, C.; Yiu, H. P.; Dobson, J. Int. J. Nanomed. 2008, 3, 169− 80. (31) Kamau, S.; Hassa, P.; Steitz, B.; Petri-Fink, A.; Hofmann, H.; Hofmann-Amtenbrink, M.; Rechenberg, B.; Hottiger, M. Nucleic Acids Res. 2006, 34, e40. (32) Nam, H.; Kim, J.; Kim, S.; Yockman, J.; Kim, S.; Bull, D. Biomaterials 2011, 32, 5213−22. (33) Han, L.; Zhang, A.; Wang, H.; Pu, P.; Jiang, X.; Kang, C.; Chang, J. Hum. Gene Ther. 2010, 21, 417−26. (34) Choi, Y. S.; Lee, J. Y.; Suh, J. S.; Kwon, Y. M.; Lee, S. J.; Chung, J. K.; Lee, D. S.; Yang, V. C.; Chung, C. P.; Park, Y. J. Biomaterials 2010, 31, 1429−43. (35) Thu, M. S.; Najbauer, J.; Kendall, S. E.; Harutyunyan, I.; Sangalang, N.; Gutova, M.; Metz, M. Z.; Garcia, E.; Frank, R. T.; Kim, S. U.; Moats, R. A.; Aboody, K. S. PLoS One 2009, 4, e7218. 2730
dx.doi.org/10.1021/bm3006903 | Biomacromolecules 2012, 13, 2723−2730
