Article pubs.acs.org/Langmuir
Retinoic Acid-Polyethyleneimine Complex Nanoparticles for Embryonic Stem Cell-Derived Neuronal Differentiation Boram Ku,† Ji-eun Kim,† Bong Hyun Chung,‡ and Bong Geun Chung§,* †
Department of Bionano Technology, Hanyang University, Ansan, Korea BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea § Department of Mechanical Engineering, Sogang University, Seoul, Korea ‡
ABSTRACT: We synthesized functional retinoic acid (RA)-polyethyleneimine (PEI) complex nanoparticles. NH groups of branched PEI chains were electrostatically interacted with carboxyl groups of RA surfaces to form cationic RA−PEI complex nanoparticles. We observed that the average diameter of RA−PEI complex nanoparticles was approximately 70 nm and the morphology of complex nanoparticles was homogeneous circular shape. To confirm the synthesis of RA−PEI complex nanoparticles, we characterized complex nanoparticles using 1H nuclear magnetic resonance (NMR), indicating that hydrophilic branched PEI chains were covered on hydrophobic RA surfaces. Furthermore, we demonstrated that pH enabled the control of amounts of RA released from RA−PEI complex nanoparticles, showing that RA exposed to acidic pH 5 was steadily released (∼76%) from complex nanoparticles, whereas RA was rapidly released (∼97%) at pH 7.4 on day 11. We also observed that RA− PEI complex nanoparticles induced embryonic stem (ES) cell-derived neuronal differentiation. Therefore, this RA−PEI complex nanoparticle is a potentially powerful tool for directing murine ES cell fate.
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INTRODUCTION Embryonic stem (ES) cell-derived neurogenesis is of great interest in regulating brain disorders and neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.1−3 The extrinsic morphogens can direct ES cell fate into specific lineages. In particular, retinoic acid (RA) plays an important role in controlling stem cell-derived neuronal differentiation. RA has been employed to regulate the neuralization and positional specification during mouse ES cell differentiation.4 It demonstrated that RA concentrations enabled the control of dorso-ventral positional identity, indicating that a dorsal and ventral phenotype was observed at higher and lower concentrations of RA. It was revealed that N-terminus of sonic hedgehog protein (Shh-N) in embryoid bodies (EBs) was up-regulated at lower concentrations of RA, suggesting the ventralization of neural progenitor cells in EBs. RA has also used to facilitate the neuronal differentiation from adherent mouse ES cells.5 It demonstrated that RA significantly enhanced neuronal differentiation of the adherent monolayer cells cultured with serum-free medium and promoted the neurite elongation (∼300 μm average neurite length) of © 2013 American Chemical Society
newborn neurons. The real-time polymerase chain reaction analysis showed that RA induced neural differentiation of mouse ES cells, confirmed by up-regulation of Wnt antagonist Dkk-1 expression. The functional nanomaterials are of great benefit for diagnostic and therapeutic applications.6−8 Various nanomaterials (e.g., nanoparticle, liposome, micelle, quantum dot, and nanotube) have been developed as molecular diagnostic probes to target the cells and tissues.8 Among biocompatible polymeric nanoparticles, polyethyleneimine (PEI)-based nanoparticles have previously been employed for applications of differentiation and nonviral gene therapy. For instance, mesoporous silica nanoparticles coated with cationic PEI polymers have been developed to regulate the cytotoxicity and differentiation of human bronchial epithelial cells.9 It demonstrated that the toxicity of epithelial cells cultured with mesoporous silica nanoparticles was directly proportional to molecular weights of Received: April 24, 2013 Revised: June 12, 2013 Published: July 11, 2013 9857
dx.doi.org/10.1021/la4015543 | Langmuir 2013, 29, 9857−9862
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Figure 1. (A) Schematic illustration of synthesis of RA−PEI complex nanoparticles and their chemical structures. (B) Transmission electron microscopy images of RA−PEI complex nanoparticles.
nanoparticles. We also confirmed that ES cells cultured with RA−PEI complex nanoparticles induced ES cell-derived neuronal differentiation.
PEI polymers. The cell surface binding of mesoporous silica nanoparticles containing PEI polymers was decreased after heparinase treatment. The poly(aspartate-g-PEI800) has been synthesized to enhance the transfection efficiency of the gene delivery.10 It showed that the polymeric length and PEI charge enabled the control of transfection efficiency and cytotoxicity, indicating that the transfection efficiency was highly increased in poly(aspartate-g-PEI800) nanoparticles containing smaller size (10 mV). It was observed that the membrane-associated toxicity was inversely proportional to polymer concentrations (20−40 μg/mL), showing that lower levels of amino groups reduced the toxicity and tissue damage. The PEI−DNA complex nanoparticles have also been used to mediate gene delivery and transfection using syndecan-1 and syndecan-2.11 The confocal image analysis showed that the endocytosis of complex nanoparticles with syndecan-1 was rapidly occurred, whereas syndecan-2 inhibited PEI-based gene transfection, suggesting contrary effects of syndecans on PEI nanoparticles-based transfection. It was revealed that the ectodomain of syndecan-2 enabled the control of the inhibitory effect on the gene transfer. Furthermore, PEIbased magnetic nanoparticles have been developed for magnetic resonance imaging and gene transfer of neural cells.12 To synthesize the functional PEI-based complex nanoparticles, Fe3O4 nanoparticles were coupled with PEI and rhodamine B isothiocyanate (RITC) were subsequently bound with DNA and polysaccharide (e.g., chitosan or dextran). The quantitative analysis represented that Fe3O4− PEI-RITC magnetic nanoparticles did not show any toxic effect on astrocytes. Although these PEI-based nanoparticles have great potential to regulate cellular behavior, RA−PEI complex nanoparticles have not yet been elucidated for directing murine ES cell fate. Therefore, we synthesized cationic RA−PEI complex nanoparticles (70 nm in diameter) and analyzed the effect of pH on RA amounts released from RA−PEI complex
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MATERIALS AND METHODS
Materials. All-trans retinoic acid (RA, 300.44 MW), branched polyethylenimine (PEI, 25,000 MW), zinc sulfate heptahydrate (ZnSO4•7H2O), dextran sulfate, dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich. All reagents were used without further purification. Synthesis of RA−PEI Complex Nanoparticles. 0.6 mL drop of RA (2% w/v in DMSO) added into 12 mL of PEI (1% w/v at pH 8.0 borate buffer), followed by stirring for 30 min. Twelve mL of dextran sulfate solution (1% w/v) was added dropwise and was subsequently stirred for 5 min. 1.2 mL ZnSO4 solution (1 M) was also added and was stirred for 30 min. Synthetic nanoparticles were centrifuged three times in 5% mannitol solution at 14,000g for 20 min. Supernatants were collected at each step. The nanoparticles were freeze-dried for 1− 3 days and were stored at −20 °C To evaluate the drug content and loading efficiency, the volume of dialyzed solution was adjusted to 25 mL. Particle Size and Zeta Potential Measurement. The sizes of nanoparticles were investigated using a dynamic light scattering (Zetasizer Marvern Instruments, France). For size distribution analysis, nanoparticles were dispersed in a deionized water and were sonicated using a sonicator (Sonics: Vibracell) at 20% amplitude for 1 min. The sample was analyzed at 25 °C and a fixed scattering angle of 90°. The zeta potential of RA, PEI, and RA−PEI complex nanoparticles was measured by laser-based multiple angle particle electrophoreses analyzer. For the zeta potential measurement, the ultrasonicated nanoparticles were placed in the electrophoretic cell with an electric field of 15.24 V/cm. Transmission Electron Microscope. The particle size and surface morphology of RA−PEI complex nanoparticles was characterized by a transmission electron microscope (TEM, JEM-2100F). An aliquot of lyophilized nanoparticles was resuspended in phosphate buffer silane (PBS). A drop of nanoparticles was suspended on a 9858
dx.doi.org/10.1021/la4015543 | Langmuir 2013, 29, 9857−9862
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Figure 2. Characterization of RA−PEI complex nanoparticles. (A) Size profile distribution of RA−PEI complex nanoparticles using dynamic light scattering. (B) Zeta-potential analysis of RA, PEI, and RA−PEI complex nanoparticles. (C) Characterization of PEI, RA, and RA−PEI complex nanoparticles using 1H NMR spectra analysis. carbon film coated on a 400 mesh copper grid and was subsequently dried overnight. 1 H Nuclear Magnetic Resonance Spectra. 1H nuclear magnetic resonance (NMR) spectra of RA, PEI, and RA−PEI complex nanoparticles were recorded by a Bruker spectrometer (400 MHz). Chemical shifts were given in ppm using tetramethylsilan (TMS) as an internal reference. 1H NMR spectra were measured using DMSO and deuterium oxide (D2O). Release Profile from RA−PEI Complex Nanoparticles. Twenty-five mg nanoparticles were placed in PBS (0.1 M, pH 7.4, 5 mL) and were incubated at 37 °C with stirring (50 rpm rate). The nanoparticle suspension was centrifuged and 4 mL of the release medium was exchanged with fresh medium to maintain the sink condition. RA released from RA−PEI complex nanoparticles was measured by a UV spectrophotometer (350 nm wavelength) and was subsequently calculated as a cumulative value. RA concentrations were calculated from the calibration curve. Murine Embryonic Stem Cell Culture. The murine ES cell (R1) were cultured on 0.1% gelatin-coated dish with Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) knockout medium supplement with 15% ES-qualified Fetal Bovine Serum (FBS, Invitrogen), 1400 U/mL leukemia inhibitory factor (LIF, Millipore), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen), 0.1 mM ß-mercaptoehanol (Invitrogen), and 1% nonessential amino acids (NEAA, Invitrogen). The culture medium was changed every day. To induce ES cell-derived neuronal differentiation, we used insulintransferrin-selenium (ITS) and N2 supplements (Invitrogen). Cellular Uptake. The cellular uptake was analyzed by fluorescein isothiocyanate (FITC)-conjugated RA−PEI complex nanoparticles using confocal microscopy. Cells (2 × 104 cells/cm2) were cultured on gelatin-coated cover glass for 24 h and were then treated FITCconjugated RA−PEI complex nanoparticles. After 18 h, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. To label cell membrane, cells were incubated in Hank’s Balanced Salt Solution (HBSS) containing wheat germ agglutinin Alexa fluor 594 conjugate
(Invitrogen) for 10 min at room temperature. After permeabilization with 1% triton X-100 for 3 min, cells were stained with DAPI (Invitrogen, USA) for 5 min at room temperature and were then mounted on a glass slide with fluorescence mounting medium. Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 15 min and were then permeabilized with 1% triton X-100 in DPBS for 3 min at room temperature. The cells were blocked with 1% BSA diluted in DPBS for 30 min at room temperature. The primary antibody was diluted in blocking buffer and was incubated overnight at 4 °C. After washing with DPBS, the Alexa Fluor 488 conjugated mouse secondary antibody (Invitrogen) was incubated for 2 h at room temperature. The cells were counterstained with DAPI (Invitrogen) for 5 min.
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RESULTS AND DISCUSSION
Synthesis of RA−PEI Complex Nanoparticles. We synthesized functional cationic RA−PEI complex nanoparticles. The cationic branched PEI polymer chains were cross-linked on anionic RA surfaces, forming RA−PEI complex nanoparticles (Figure 1A). For RA−PEI complex nanoparticles, carboxyl groups (−COOH) of hydrophobic RA was electrostatically interacted with NH groups of hydrophilic PEI to generate RAPEI complex nanoparticles with a positive net charge. TEM images showed that the morphology of synthesized RA−PEI complex nanoparticles was uniform and homogeneous circular shape (Figure 1B). It was revealed that the surface of RA−PEI complex nanoparticles was rough, showing that branched PEI polymer chains covered RA surfaces. Characterization of RA−PEI Complex Nanoparticles. To characterize the size and charge distribution of RA−PEI complex nanoparticles, we employed the dynamic light scattering and zeta-potential. The analysis of the dynamic 9859
dx.doi.org/10.1021/la4015543 | Langmuir 2013, 29, 9857−9862
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7.4 and pH 5 was similar for 3 days, whereas we found the significant discrepancy of cumulative release at day 4−11. It was revealed that 90% cumulative release of RA was observed at pH 7.4 on day 4, following steady increase for 11 days (∼97%). In contrast, we found 53% cumulative release of RA from RA−PEI complex nanoparticles at pH 5 on day 4, following steady increase for 11 days (∼76%). It has been known that RA was rapidly released, as previously described.13 Previous study suggested that the rapid release behavior of the hydrophobic RA would be inhibited by the hydrophilic materials. Similar to the previous study, we also demonstrated that hydrophilic PEI enabled the control of the release of hydrophobic RA. Neuronal Differentiation from Murine Embryonic Stem Cells. Cellular uptake of nanoparticles played an important role in inducing ES cell-derived lineage commitments. We observed that murine ES cells uptaked RA−PEI complex nanoparticles (Figure 4). Confocal microscopic images
light scattering represented that the size of RA−PEI complex nanoparticles was approximately 70 nm (Figure 2A). We also measured the charge of RA−PEI complex nanoparticles, indicating that RA showed a negative net charge, whereas PEI and RA−PEI complex nanoparticles were cationic (Figure 2B). For the zeta-potential analysis, we used RA (7.4% w/w) in complex nanoparticles and 96% w/w loading efficiency. Zetapotential analysis revealed that PEI played an important role in determining the charge of RA−PEI complex nanoparticles. Interestingly, we observed the narrow range of cationic PEI and RA−PEI complex nanoparticles compared to the wide range of anionic RA, because PEI covered surfaces of complex nanoparticles. To confirm the chemical structures whether RA was cross-linked with PEI for creating RA−PEI complex nanoparticles, we analyzed RA−PEI complex nanoparticles using 1H NMR spectra (Figure 2C). It showed that the pure hydrophilic PEI had two major peaks (a, b), whereas the pure hydrophobic RA showed several peaks (c−l). For pure PEI, peaks a and b indicated the carbon chain and NH group, respectively. We observed the RA components, showing that peaks h−o represented carboxyl group (COOH−). Interestingly, although the pure RA showed the carboxyl group, we did not observe carboxyl group in the RA−PEI complex nanoparticles, because the carboxyl group of RA was cross-linked into NH group of PEI to form amide group (C−O−N). For RA−PEI complex nanoparticles, we observed two major peaks (a, b) and several minor peaks (h−o). It revealed that the peaks (c−g, j, n) of the hydrophobic RA were removed in RA−PEI complex nanoparticles, indicating that the surface property of RA−PEI complex nanoparticles was hydrophilic. As a result, NMR analysis demonstrated that the surface of RA−PEI complex nanoparticles was covered by hydrophilic PEI. Controlled Release of RA from Complex Nanoparticles. RA−PEI complex nanoparticle is of great benefit for regulating the drug release in a controlled manner. We hypothesized that the cumulative release of RA would be controlled by pH and hydrophilic RA−PEI complex nanoparticles would enable the control of the cumulative release of the hydrophobic RA. We analyzed whether RA was released from RA−PEI complex nanoparticles in a controlled manner (Figure 3). Although RA was slightly released from RA−PEI complex nanoparticles (
