In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis.
RNA interference (RNAi), a powerful technology for sequence-specific gene silencing, has been widely used in silencing many important oncogenes that play major roles in various stages of tumor development including cell transformation, unrestricted tumor cell proliferation and metastasis1,2. However, efficient delivery of small interfering RNA (siRNA) to targeted sites remain a major hurdle to overcome owing to the rapid degradation by serum nucleases, hepatic clearance, low transfection efficiency, off-target effect, and inefficient release from endosomes2,3. Generally, there are two major nucleic acid delivery systems, viral and nonviral systems. The viral vector generally has high efficiency in delivery, but the lack of tissue/cell targeting ability, cytotoxicity, immunogenicity, and limited capacity to accommodate large inserted genetic materials should be considered when used in vivo. Non-viral nano-carrier gene delivery systems, such as liposomes, nano-hydrogel, biodegradable nanoparticles, and inorganic nanoparticles, generally have lower transfection efficiency compared to their viral counterparts. However, because these non-viral nano-carriers possess low immunogenicity, low cytotoxicity, ease of manipulation, their transfection efficiency and formulations have been significantly optimized to meet the requirements for gene delivery. Although siRNA can now be easily delivered into cultured cells by viral or non-viral approaches, targeted delivery in vivo remains a big challenge in cancer gene therapy. Inorganic nanoparticles such as Fe3O4 may become an ideal siRNA carrier in vivo owing to its nano-sized particles for crossing physiological barriers and its potential for various surface modifications that can be used for targeted delivery.
In the last two decades, there has been an ever-increasing interest in fabricating nanometer-scale carriers for drug or gene delivery. Among them, superparamagnetic iron oxide nanoparticle (SPIO), for instance, Fe3O4 nanoparticle, is a promising candidate for their unique magnetic property, large surface area, and low cytotoxicity. However, two limitations need to be surpassed before they can be used as an efficient drug/gene delivery system. One is few functional groups on their surfaces, and the other is low dispersity and easy to aggregate or deposit. These limitations could largely be overcome by surface coating of the magnetic nanoparticles. Silica is one of the exceptional coating materials for encapsulating magnetic nanoparticles because of its good biocompatibility, excellent physicochemical stability, and its ease of surface multi-functionalization. In general, silica coating can be achieved by the Stöber process or the reverse (water-in-oil) microemulsion method3,4,5,6. These silica-coated fluorescent magnetic nanoparticles have been widely used to develop biomedical platforms for simultaneous imaging, diagnosis, and therapy. However, fabricating a desirable outer surface that can be further modified by adding another functional group or a targeting moiety remains challenging.
For nucleic acid, for instance, siRNA delivery, the silica surface charge should be positive. Introducing cationic charge on inorganic materials typically involve surface grafting with amine groups and coating with cationic polymers through either covalent or electrostatic association. These positively charged nanoparticles have been demonstrated to be able to carry nucleic acids and can be used an alternative to traditional viral vectors7. Polyethyleneimine (PEI), which contains primary, secondary, and tertiary amines to complex with nucleic acids such as DNA or siRNA, is one of the most widely studied cationic polymers for gene delivery. In addition, PEI can be easily modified by coupling some small molecules to endow it specific targeting ability. PEI can also induce cytotoxicity at high molecular weight and in high concentration, which is usually not the case in its application as a nanoparticle carrier8.
Efficient targeted delivery of therapeutics is an important aspect to consider when designing targeting nanoparticles for cancer gene therapy. Insufficient uptake by targeted tissues will decrease the therapeutic benefit and affect the quality of imaging, which at the same time may increase the concentration of the therapeutics at the off-target sites and thus increasing the toxic side effects. One way to increase the local concentration of nanotherapeutics in tumor tissue is to conjugate these nanoparticles with targeting molecules that have high affinity to targeted tumor cells9. Folic acid (FA), a nonimmunogenic ligand, has emerged as an attractive specific targeting molecule for anticancer drug delivery because folate receptors are often overexpressed on the surface of many human cancers including ovarian, lung, breast, endometrial, renal, and colon cancers10,11,12,13. This folate receptor-mediated targeting has also been used as an imaging probe for cancer diagnosis14. Folate ligand is expected to gain popularity as a targeting molecule in tumor theranostics because of its simple and well-defined conjugation chemistry.
Accumulating evidence showed that Notch signal pathway played a key role in normal development and was increasingly recognized for its importance in cancer biology14,15. In mammalian, Notch pathway includes four transmembrane receptors (Notch-1 to 4) and five ligands (Delta-like 1, 3, 4, Jagged 1, 2). Upon activation, Notch-1 was cleaved by γ-secretase and released the Notch-1 intracellular domain (NICD) from the plasma membrane. NICD then translocates into the nucleus of the cells, where it formed a complex with transcriptional factors that regulated the expression of target genes and leading to altered cellular behaviors. Aberrant Notch signaling has been demonstrated to facilitate the proliferation and survival of tumor cells by regulating downstream effectors or other signaling pathways. Hence, targeting Notch signaling pathway is a promising treatment option for cancer.
In this study, a magnetic and fluorescent core/shell nanoparticles-based Notch-1 shRNA delivery system (denoted as Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA) was created, where FA was conjugated to PEI to increase its targeting capacity. The physiochemical characteristics of the Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex were analyzed by agarose gel electrophoresis, nuclear magnetic resonance spectra, and zeta potential measurements. The in vitro cytotoxicity, biocompatibility, cellular uptake efficiency, intracellular biodistribution, and the effects on cell proliferation and apoptosis in human breast cancer MDA-MB-231 cells upon treatment with the nanocomplex were extensively evaluated. We found this new sub-100 nm nanocomplexes exhibited low cytotoxicity, excellent colloidal stability in dilute aqueous dispersions, good biocompatibility, the ability to shield the loaded shRNA from nuclease degradation, and efficient targeted delivery of Notch-1 shRNA. We further demonstrated that the fabricated multifunctional Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex possesses magnetic properties that could be used as magnetic imaging probes at cellular level. The multifunctional nanocomplex has the potential to be exploited as a theranostics for targeted cancer gene therapy and MR imaging for cancer diagnosis.
Materials and chemicals
Tetraethylorthosilicate (TEOS), 3-aminopropyltrimethoxysilane (APS), fluorescein isothiocyanate (FITC), branched polyethyleneimine (PEI, 25 kD), N,N′-Dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (St Louis, MO, USA). Iron oxide nanoparticles (10 nm) were obtained from Nanjing Emperor Nano Material Co, Ltd (Nanjing, China). L15 cell culture medium, fetal bovine serum (FBS), and trypsin were purchased from Gibco (Grand Island, NY, USA). Folic acid (FA) was obtained from Alexis (Los Angeles, CA, USA). Trizol and calcein-AM were from Invitrogen (Carlsbad, CA, USA). Rabbit anti-human Notch-1 polyclonal antibody was purchased from Cell Signaling Technology (Boston, MA, USA). All chemicals were used as received without further purification.
Synthesis of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex
Fe3O4@SiO2(FITC) was prepared following a previously described procedure with some minor modifications4,16,17. Briefly, 2 mg of FITC was mixed with 125 μl of APS in 1 mL of anhydrous ethanol in the dark and allowed to react at room temperature for 24 h. The resulting mixture of FITC-APS conjugates in solution was then stored at 4°C. One milliliter of aqueous solution containing magnetic particles was diluted with 1 mL of water and 7.5 mL of anhydrous ethanol. Fifty microliters of TEOS and 200 μl of ammonia solution (28–30% by NH3) were added to this reaction mixture containing magnetic particles while stirring at 300 rpm for 24 h at room temperature, followed by FITC-APS solution addition with continuous stirring for 48 h. The reaction was terminated by adding methanol. The Fe3O4@SiO2(FITC) nanoparticles were harvested by centrifugation at 12,000 rpm for 5 min and washed five times with deionized water to remove excess reactants.
To noncovalently couple Notch-1 shRNA to the surface of the Fe3O4@SiO2(FITC) nanoparticles, co-polymer of PEI-FA were synthesized as follows: FA was activated in the presence of DCC and NNS by gentle stirring (300 rpm) for 12 h at room temperature (25°C), followed by addition of PEI to allow for another 12 h gentle stirring. The reaction mixture was then dialyzed for 48 h against deionized water to remove any unreacted reactants. The PEI-FA solution was added to the Fe3O4@SiO2(FITC) nanoparticle suspension (1 ml), which were then mixed by rotating for 2 h at a speed of 180 rpm to allow PEI-FA to graft onto the surface of Fe3O4@SiO2(FITC) nanoparticles. Unbound PEI-FA was removed by centrifugation at 10,000 rpm for 5 min with deionized water for three times. Notch-1 shRNA was incubated in the Fe3O4@SiO2(FITC)/PEI-FA suspension for 30 min at room temperature to form Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by electrostatic absorption at various weight ratios. The synthetic procedure for the Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex is summarized in Figure 1.
Characterization of nanocomplex
The size and morphology of the nanoparticles were examined by transmission electron microscopy (TEM) (JEM-100CX, JEOL, Tokyo, Japan) and scanning electron microscopy (SEM) (JSM-6490LV, JEOL, Tokyo, Japan). An aqueous dispersion of the nanoparticles was drop-cast onto a carbon-coated copper grid, and the grid was air-dry at room temperature before loading into the microscope. The presence of Fe3O4 and silica shell in the nanoparticles was evidenced by energy-dispersive X-ray (XRD) analysis. The zeta potential and the particle size distributions were determined using laser dynamic light scattering with Mastersizer 2000 (Malvern Instruments, Malvern, UK) in water at 25°C in accordance with the manufacturer's operating manual. The presence of FITC doped into the silica shell was confirmed using an inverted fluorescence microscope (TE-2000U, Nikon, Tokyo, Japan).
1H NMR study
The covalent bonding between PEI and FA was ascertained using a VARIAN 400-MHz 1H NMR spectrometer (Varian Inc, Palo Alto, CA). The lyophilized product was dissolved directly in D2O. PEI and FA were also dissolved in D2O. The production of new bonds was evaluated by the chemical shift of 1H protons of benzene ring of FA in the range of 6.5–9.0 ppm.
Magnetic measurement of samples was performed on a superconducting quantum interference device (SQUID) magnetometer (MPMS XL-7, Quantum Design, San Diego, CA, USA). For measurement, about 10 mg powder of the sample was inserted in a gelatin capsule at room temperature. The magnetic hysteresis loop was measured at room temperature.
Gel retardation assay
To test the absorption ability of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles to Notch-1 shRNA, Notch-1 shRNA was incubated in the Fe3O4@SiO2(FITC)/PEI-FA suspension for 30 min at room temperature to form Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by electrostatic absorption at the weight ratios of 5:1, 10:1, 20:1, and 30:1. The nanocomplex was confirmed by electrophoresis on a 1% agarose gel with tris/acetate/EDTA buffer at 100 V for 60 min, and visualized by staining with ethidium bromide. The images were acquired using a UV transilluminator (Bio-Rad, Philadelphia, PA, USA).
Notch-1 shRNA stability assay
For the protective effect assay, naked Notch-1 shRNA or Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex was incubated with DNaseI (1 U/μl) for 30 min. For stability assay in cell culture medium, Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex at the weight ratio of 20:1 or naked Notch-1 shRNA was incubated in L15 culture medium containing 10% FBS for 12, 24, and 48 h. Then treated samples were loaded onto a 1% agarose gel containing 0.01% ethidium bromide and run at 100 V for 60 min. Notch-1 shRNA was released from Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by treatment with 1% sodium dodecylsulfate (SDS).
Cell viability assay
Cells were plated at 8000 cells per well in 100 μl of culture medium on 96-well plates. Twenty-four hours after plating, 10, 20, 40 and 80 μg/ml of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles were added to the cells, in triplicate at each concentration, and incubated for 24, 48, and 72 h, respectively. Cell viability was evaluated using the WST-8 cell counting kit (Beyotime, Beijing, China) in accordance with the manufacturer's instructions as previously described18. The absorbance was recorded on a microplate reader (Model 680, Bio-Rad, Philadelphia, PA, USA) at a wavelength of 450 nm. Data were expressed as a ratio of treated to untreated cells (control).
Blood from healthy SD rat was collected in heparin-coated tubes as described previously with minor modifications19,20. The serum was removed from the blood by centrifugation at 1500 rpm for 5 min, and the red blood cells (RBCs) were then washed three times with sterile isotonic 0.9% NaCl solution. Following the last wash, the RBCs were resuspended with sterile isotonic 0.9% NaCl. The RBC suspension (300 μl) was mixed with 1.2 ml of 10, 20, 40 and 80 μg/ml of Fe3O4@SiO2(FITC)/PEI-FA nanoparticle suspension respectively. Positive and negative controls were also prepared by mixing 300 μl of RBC suspensions with 1.2 ml of deionized water (H2O) and 0.9% NaCl, respectively. After incubation for 2 h, the samples were then centrifuged for 2 min at 4000 rpm. The absorbance of the supernatant was recorded at 541 nm UV-vis spectroscopy (Hitachi UV-2910, Japan), which is directly proportional to the amount of hemoglobin released.
Evaluation of cellular internalization
MDA-MB-231 cells were seeded on coverslips in a 6-well plate at a seeding density of 5 × 105 cells/well, and the medium was refreshed after 24 h. After treatment with Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)/PEI, Fe3O4@SiO2(FITC)/PEI-FA or Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles, the cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), and then fixed with 4% paraformaldehyde solution in PBS for 15 min at 37°C. The coverslips were then washed three times with PBS to remove nonspecific binding. The cells were further treated with DAPI for 15 min. For FA competition experiments, free FA (1 mM) was preincubated with MDA-MB-231 cells for 2 h prior to addition of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles. All the samples were examined under either an inverted fluorescent microscope (Nikon TE-2000U, Japan) or a confocal laser scanning microscope (TCS SP5, Leica, Germany).
Magnetically targeted delivery assay
MDA-MB-231 cells were seeded in 35-mm culture dishes at 3 × 105 cells per well one day before the experiment. On the second day, 100 μg of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles was added to each dish. In vitro magnetically targeted delivery was conducted by placing a magnet under the center of the cell culture dish21. After 6 h incubation, the cells were washed three times with sterile PBS. The cellular uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex was observed under inverted fluorescent microscope (Nikon TE-2000U, Japan). For cell apoptosis analysis with an external magnetic field, the cells were continued to culture for another 72 h, and then calcein-AM/PI double-staining was performed as described in “Cell apoptosis assay” section.
Total RNA isolation and RT-PCR
Total RNA was isolated from cultured MDA-MB-231 cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions22. One microgram of total RNA from each sample was used for reverse transcription in a 20(μl reaction volume following vendor's instructions. One microliter of each cDNA was used for PCR in a 25(μl reaction. PCR cycling conditions were 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. PCR products were resolved on 1% agarose gel with ethidium bromide and the bands were visualized under ultraviolet illumination. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used for PCR were: Notch-1 (forward, 5(-GGAGCATGTGTAACATCAACA-3′; reverse, 5&prime-CCTCGTACACATTGTAGTT GT-3&prime); and GAPDH (forward, 5&prime--GTCTCCTCT GACTTCAACAGCG-3′; reverse, 5&prime--ACCACCCTGTTGCTGTAGCCAA-3&prime)).
Western blot analysis
After treatment with aforementioned nanoparticles (50 μg/ml), cells were harvested, washed twice with ice-cold PBS, and lysed in a lysis buffer (Beyotime, Beijing, China) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100 and several protease inhibitors. Total protein concentrations were determined by a bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). For Western blot analysis23, an equal amount of lysate (20 μg) was resolved on 10% SDS-PAGE and electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with PBST containing 5% skim powder milk for 1 h at room temperature, and then incubated with rabbit anti-human Notch-1 polyclonal antibodies at 1:1000 for 1 h at room temperature. After washing, the membranes were further probed with horseradish peroxidase-conjugated goat anti-rabbit IgG at 1:5000 dilution. The blots were developed using chemiluminescence (BeyoECL Plus) (Beyotime, Beijing, China).
Cell proliferation assay
For cell proliferation assay, MDA-MB-231 cells were plated in a 96-well plate at a density of 8 × 104 cells/well and grown overnight. Nanoparticles (10 μg/ml) were added to the cells and continued to incubate for 6 h, washed three times with warm PBS (37°C), and growth medium were refreshed. Cell proliferation was measured by WST-8 assay at 24, 48 and 72 h. All experiments were performed in triplicate. Data were expressed as ratio of treated to the untreated cells (control).
Cell apoptosis assay
Calcein-AM/PI double-staining was used to evaluate apoptotic cell death (red)24. MDA-MB-231 cells were seeded in six-well plates (2 ml, 5 × 105 cells/well) and incubated overnight at 37°C to allow the cells to adhere. After treatment with 50 μg/ml aforementioned nanoparticles for 6 h, cells were rinsed with PBS for three times and continued to culture for 72 h. The cells were then stained by a mixture of calcein-AM and PI solution for 10 min. Apoptotic cell death (red) were examined under an inverted fluorescent microscope (Nikon TE-2000U, Japan).
Cellular MR imaging
The day before the experiment, MDA-MB-231 cells were seeded onto 12-well plates at a density of 2 × 105 cells per well. Twenty-four hours after seeding, nanoparticles at a concentration of 25.6 μg/ml were added, and a saline solution without any nanoparticles was used as the control. After 6 h incubation, the cells were washed five times with PBS to remove the non-internalized nanoparticles, harvested, and resuspended in 1 mL PBS for MRI. Fe3O4@SiO2(FITC)/PEI-FA nanoparticles were re-suspended in the cell suspension at iron concentration of 1.6, 3.2, 6.4, 12.8, and 25.6 μg/ml, respectively, and MR imaging was performed at 25°C in a clinical 3.0 Tesla Clinical Siemens Trio scanner (Discovery MR750, GE Healthcare, Boulder, CO, USA). T2-weighted images were acquired using spin-echo imaging sequences.
All experiments were carried out at least in triplicate. Data were presented as the mean ± standard error of the mean (SEM). The statistical analysis was performed using GraphPad Prism Software version 5.0 (GraphPad Software Inc., San Diego, CA, USA). Statistical significances were determined using the One-way ANOVA followed by Bonferroni tests, and p < 0.05 was considered statistically significant.
Results and Discussion
Synthesis and physicochemical characterization of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex
The synthetic route for PEI-FA coated Fe3O4@SiO2 nanoparticles is shown in Figure 1. The oleic acid-stabilized Fe3O4 nanoparticles were first coated with TEOS and APS-FITC to form a fluorescent silica shell. PEI-FA was then electronically absorbed to the surface of Fe3O4@SiO2(FITC) nanoparticles to enable Fe3O4@SiO2(FITC) nanoparticles to target cancer cells because folate receptor is overexpressed in a wide variety of tumors including breast, lung, and cervical cancer15,25,26. We have previously shown that FA provides a good cancer cell-specific target in vitro27. The modification of Fe3O4@SiO2 presented here also leads a high positively charged surface of the nanoparticles, which enable to load more negatively charged DNA or RNA by electronic absorption. Furthermore, an improved microemulsion method was employed to create the Fe3O4@SiO2(FITC)/PEI-FA nanoparticles for gene delivery.
Scanning electron observations revealed that the Fe3O4@SiO2(FITC) nanoparticles were uniform and averaged 64 nm in diameter (Figures 2A), and that PEI-FA modification and Notch-1 shRNA complexation did not affect the morphology and size distribution of the nanoparticles, but showed a slight tendency to aggregate in solution (Figures 2B and C), which might be caused by the increased surface charge of the particles and Notch-1 shRNA cross-linking. The Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles could be briefly treated with ultrasonication under acidic solution condition to reduce the aggregation of particles (data not shown). TEM indicated that the magnetic nanocrystal embedded in silica retained its original crystallinity after silica coating and surfactant extraction (Figure 2A, insert). The uniform silica coating of the Fe3O4 nanocrystal was further confirmed by EDX method (Figure 2D). The presence of silica and iron in the nanocomposite indicates that core/shell Fe3O4@SiO2 structure is formed. Compared to the Fe3O4@SiO2(FITC) nanoparticles (See Figure S1 in supporting information), the Fe3O4@SiO2(FITC)/PEI-FA nanoparticles have a better dispersity in PBS at room temperature as Fe3O4@SiO2(FITC)/PEI-FA nanoparticles remained well dispersed after 12 h, suggesting that the surface modification of Fe3O4@SiO2(FITC) with PEI-FA improved their dispersity. Furthermore, all the nanoparticles moved to the bottom of the tube soon after the magnet was placed, which also demonstrated that the core-shell nanoparticles were successfully synthesized, and the resultant nanocomposites can be manipulated by an external magnetic field.
FITC-APS conjugates were prepared in advance via an addition reaction between the isothiocyanate group of FITC dye and the primary amine group of APS. This synthetic process enables the co-encapsulation of Fe3O4 nanoparticles and a large number of FITC dye molecules to stay together within the silica shell4,28,29. Using fluorescence microscopy, we confirmed that both Fe3O4@SiO2(FITC) and Fe3O4@SiO2(FITC)/PEI-FA nanoparticles were well-dispersed, and distinct fluorescence existed for the two nanoparticles (Figure 3). It indicated that PEI-FA modification had no obvious influence on fluorescence intensity (Figure 3B). The 1H NMR analysis was used to determine the composition of the resulting polymers. In the 1H-NMR spectrum of PEI-FA in D2O as shown in Figure 4A, the signals at δ 2.0–3.0 ppm were attributed to the methylene group in PEI, and the signals at δ 6.5–9.0 related to the H of benzene ring in FA30,31. The results indicate that about one FA molecular chain was conjugated to one PEI molecular chain. To verify whether the PEI-FA co-polymer were coated on the surface of Fe3O4@SiO2(FITC) nanoparticles, fluorescence emission spectra were used and the results showed an emission peak at the wavelength of approximately 440 nm for the Fe3O4@SiO2(FITC)/PEI-FA nanoparticle suspension (Figure 4B), which is the characteristic fluorescence emission peak of FA4,10,31, suggesting that PEI-FA molecules were successfully coated onto the surface of Fe3O4@SiO2(FITC) nanoparticles. The zeta potential analysis further showed a positive surface charge of +17.5 ± 1.3 mV (Figure 4C) for the Fe3O4@SiO2(FITC)/PEI-FA nanoparticle, suggesting that the Fe3O4@SiO2(FITC)/PEI-FA nanoparticle could bind to the negatively charged Notch-1 shRNA through electrostatic interaction. For assessment of the magnetic properties and sensitivity of the formulated nanoparticles, magnetic hysteresis loops were recorded using a magnetometer, and the three types of nanoparticles showed superparamagnetic behavior without magnetic hysteresis (Figure 5) at room temperature (about 300 K). They were found to have no coercive fields, thereby confirming their superparamagnetic nature. There was also no significant difference in their magnetic hysteresis loops and magnetization saturation values even when the Fe3O4@SiO2(FITC)/PEI-FA nanoparticles were modified with the Notch-1 shRNA.
Biological characterizations of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex
The nuclear acid stability and nuclear acid-binding capability of the nanoparticles are two very important factors to consider in choosing a gene delivery carrier32,33. The condensed form of the nanoparticles/gene complex can protect the nuclear acid from degradation when they pass through cell barriers during gene delivery. In order to study shRNA stability and condensation capabilities of nanoparticles with shRNA, agarose gel electrophoresis was performed. Complexation of nucleic acids with cationic polymers is driven by electrostatic neutralization, during which nucleic acids partially or completely loses negative charge and, consequently, loses mobility in electric field30,34. Therefore, by observing he motility of Notch-1 shRNA in gel electrophoresis one can determine Fe3O4@SiO2(FITC)/PEI-FA ability to complex with Notch-1 shRNA. With the increasing of NPs/pDNA weight ratio from 5:1 to 30:1, the electrophoretic mobility of the Notch-1 shRNA gradually retarded, indicating the enhanced binding of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles to Notch-1 shRNA. When the NPs/shRNA weight ratio reached 15:1, no free Notch-1 shRNA bands were observed on the gel, indicating that Notch-1 shRNA had completely complexed with the Fe3O4@SiO2(FITC)/PEI-FA nanoparticles (Figure 6A). We also used DNaseI to further investigate the stability and protection of NPs/shRNA. We found that naked Notch-1 shRNA was completely degraded by DNaseI within 30 min, while Notch-1 shRNA retained in the gel wells by Fe3O4@SiO2(FITC)/PEI-FA binding showed no signs of DNaseI degradation (Figure 6B), demonstrating that PEI-FA could concentrate and wrap Notch-1 shRNA to protect it from exposure to these agents such as nucleases or other destructive enzymes. Moreover, when naked Notch-1 shRNA or Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex (NPs/shRNA) were incubated in L15 cell culture medium containing 10% serum from 12 to 48 h, naked Notch-1 shRNA was completely degraded and could not be visualized in the agarose gel, whereas NPs/shRNA nanocomplex could still be visualized in the gel even after incubation for 48 h, demonstrating that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex could protect Notch-1 shRNA from serum degradation. Taken together, these results proved that the Fe3O4@SiO2(FITC)/PEI-FA nanoparticles can be used to as an efficient gene carrier.
For the ultimate use of the nanoparticles as gene carrier, it is critical that these nanoparticles after coating with silica and PEI-FA retain their low toxicity, i.e., the gene carrier should not induce cytotoxic effects35,36,37. In order to evaluate the cytotoxicity of resulting nanoparticles, the viability of human breast cancer MDA-MB-231 cells was tested in the presence of Fe3O4@SiO2(FITC)/PEI-FA, Fe3O4@SiO2(FITC)/PEI-FA/scrambled shRNA, or equal volume of cell culture medium as a control. As shown in figure 7, both Fe3O4@SiO2(FITC)/PEI-FA and Fe3O4@SiO2(FITC)/PEI-FA/scrambled shRNA nanoparticles showed no significant cytotoxicity compared to control cells. To better define the biocompatibility of the nanocomplex as gene carrier, a hemolysis assay was conducted to evaluate its compatibility in blood. During the hemolysis assay, hemoglobin released into the solution is direct proportional to hemolytic activity of the carrier, therefore the absorbance 541 nm can be used to quantify the hemolytic activity20. As shown in Figure 8, no visible hemolysis can be observed for Fe3O4@SiO2(FITC)/PEI-FA nanoparticles at 80 mg/ml. Quantitative analysis by UV-vis spectroscopy also showed that Fe3O4@SiO2(FITC)/PEI-FA nanoparticles had negligible hemolytic activity.
Cellular uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex into human breast cancer cells
In vitro cellular uptake experiments were performed using human breast carcinoma MDA-MB-231 cells, known to significantly overexpress the folate receptor15,25,26. The uptake of the nanoparticle conjugates was evaluated after 6-h cell culture, and is shown in Figure 9. In contrast to Fe3O4@SiO2(FITC) or Fe3O4@SiO2(FITC)/PEI, the uptake of FA-conjugated Fe3O4@SiO2(FITC)/PEI-FA by MDA-MB-231 cells was ~3-fold higher than those cultured with Fe3O4@SiO2(FITC). PEI, a cationic polymer, could also facilitate nanoparticle uptake in some extent. While the increase in intracellular Fe3O4@SiO2(FITC)/PEI nanoparticles was somewhat visible, however, the fluorescent intensity was considerably less than that in FA-conjugated Fe3O4@SiO2(FITC)/PEI-FA nanoparticles. Furthermore, high uptake was also observed in Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex (Figure 10), which demonstrated that absorption of Notch-1 shRNA to the nanoparticles did not affect uptake. To ascertain that the nanoparticles were indeed internalized rather than being bound to cell membrane, MDA-MB-231 cells incubated with various nanoparticles were examined by confocal fluorescence microscopy. As shown in Figure 10, the nanoparticles mainly localized to the cytoplasm after 6 h culture. These results agreed well with that of inverted fluorescence microscopy, and also confirmed that the uptake of FA-conjugated Fe3O4@SiO2(FITC)/PEI-FA nanoparticles was more favorable than other non-FA-conjugated nanoparticles.
The specific targeting of the folate receptor by Fe3O4@SiO2(FITC)/PEI-FA nanoparticles was further verified by a competitive inhibition assay, in which MDA-MB-231 cells were pretreated with free FA before culturing with Fe3O4@SiO2(FITC)/PEI-FA nanoparticles. As expected, a sharp decreased uptake of the nanoparticles by MDA-MB-231 cells was observed (Figure 10). This result indicates that the uptake of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles was mediated by folate receptor on the cell surface9,38. Taken together, these findings indicated that conjugation of Fe3O4@SiO2(FITC) nanoparticles with a tumor targeting agent, FA specifically, led to the increased targeted uptake of Fe3O4@SiO2(FITC)/PEI-FA nanoparticles by MDA-MB-231 cells bearing FA receptor.
Silencing Notch-1 gene expression and anti-tumor effect of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex
To evaluate the Notch-1 gene silencing effect of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex, human breast carcinoma cells (MDA-MB-231) were incubated with the nanoparticles as described in materials and methods. As shown in Figure S2 (see Supporting Information), Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex efficiently decreased the expression of Notch-1 at both mRNA and protein level in MDA-MB-231 cells compared to that in nanoparticles containing either a scrambled shRNA or untransfected cells. These results suggest that the Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex could silence Notch-1 expression in a highly sequence-specific fashion39,40.
We then investigated whether these nanoparticles have any effect on cell proliferation. Cells were treated with various nanoparticles for 24, 48 and 72 h, and it showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex inhibited MDA-MB-231 cell proliferation, especially at 48 and 72 h (Figure 11). To evaluate whether the inhibition of cell proliferation by Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex was due to its apoptotic effect, the cell apoptosis was evaluated by calcein-AM/PI double staining. As shown in Figure 12, more cell apoptotic cells were observed in cells treated with Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex compared to control treatment. These data suggest that the inhibited cell proliferation induced by Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex was at least partially attributed to its apoptotic effect. Notch-1 signaling pathway is an evolutionarily conserved signaling pathway that has been shown to regulate many cellular processes including cell proliferation, differentiation, apoptosis, and survival41,42. In light of the previous finding that silencing Notch-1 promoted cancer cell apoptosis24,42, our data indicate that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex can be used as an efficient gene carrier for targeted killing of cancer cells bearing folate receptor.
Besides the folate receptor-mediated molecular targeting, the magnetic property of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex could be also utilized for magnetically targeted delivery. When MDA-MB-231 cells were incubated with Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex for 6 h, instead of 72 h for apoptosis assay, in the presence of a magnetic field under the center of the cell culture dish (Figure 13A), the uptake of the nanoparticles (green cells) by cells near the magnet was significantly higher than those far from the magnet (Figure 13B). Accordingly, the cells near the magnet showed apoptotic phenotype (red) compared to those far from the magnet (Figure 13C). Collectively, our data demonstrated that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex is an efficient gene delivery system possessing both molecular targeting and magnetic targeting capabilities.
MR imaging of cells after nanoparticle internalization
To evaluate the potential of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex to be used as a targeted MR contrast agent, MDA-MB-231 cells were cultured with Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)/PEI, Fe3O4@SiO2(FITC)/PEI-FA or Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA as described in materials and methods. Cells were harvested and resuspended in PBS and the T2-weighted MR phantom images were obtained. As shown in Figure 14, cells incubated with Fe3O4@SiO2(FITC)/PEI-FA or Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA showed a significant negative contrast enhancement (signal darkening) over other cells. As the concentration of the Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex increases, the T2-weighted MR image becomes darker for MDA-MB-231 cells, suggesting that the nanocomplex could also be used as a probe for T2 MR imaging. Our data demonstrated that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex can be exploited as a novel theranostic for cancer gene therapy and imaging probe for cancer diagnosis.
In summary, a multifunctional Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex with specific targeting ability is developed. The multifunctional nanocomplex was stable and dispersed in aqueous solution, and showed minimal cytotoxicity, and possesses dual functions as both a gene delivery vehicle and a MR imaging contrast agent. The cellular uptake of the nanocomplex was significantly enhanced in the folate receptor overexpressed human breast cancer cell lines (MDA-MB-231). Moreover, the cell proliferation treated by Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex was significantly inhibited, contributing to the increased intracellular delivery of Notch-1 shRNA and subsequently down-regulating the Notch-1 expression to inhibit the Notch-1 signal pathway. Although the in vivo therapeutic effects should be further investigated, based on the in vitro data and findings, Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex presented here may be exploited as a theranostic candidate for human breast cancer gene therapy and diagnosis.
We would like to thank the financial supports, in whole or in part, by the National Natural Science Foundation of China (11272083, 81201192, 81101147, 31470906, 31470959, 81471785), the Sichuan Youth Science and Technology Foundation of China (2014JQ0008, 2010JQ0004), the Postdoctoral Program of China (2011M501297, 2012T50715), and the Fundamental Research Funds for Central Universities in China (ZYGX2010X019, ZYGX2010J101, ZYGX2011J099).
Figure S1 and S2
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Antibody conjugated onto surface modified magnetic nanoparticles for separation of HER2+ breast cancer cells
Journal of Magnetism and Magnetic Materials (2019)