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Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle–cell interaction Yi-Ching Lu,ae Pei-Chun Luo,a Chun-Wan Huang,a Yann-Lii Leu,b Tzu-Hao Wang,c Kuo-Chen Wei,d Hsin-Ell Wange and Yunn-Hwa Ma*a Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1–3 mM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 C, but not when EGCG was removed from the
Received 2nd February 2014 Accepted 24th June 2014
culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP–EGCG composite produced by adsorption of EGCG and magnetic separation also led to an
DOI: 10.1039/c4nr00617h
enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system,
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which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.
Introduction In targeted delivery of therapeutic agents, a targeting carrier is expected to increase drug concentration at the target site and thus reduce systemic distribution/side effects. A magnetic nanoparticle (MNP), composed of an iron oxide core and a polymer coating, has been tested in animal models as a magnetically guided carrier in the delivery of chemotherapeutic1–3 or thrombolytic agents.4,5 In such biomedical applications, the interaction of cells and nanoparticles is crucial for the pharmacological efficacy of the carrier-drug system, depending on the sites of the target. In targeted chemotherapeutics, MNP internalization by tumor cells may determine the therapeutic a
Department of Physiology and Pharmacology, Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, Republic of China. E-mail: [email protected] b
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, Republic of China
c Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, 5 Fusing St., Kwei-Shan, Tao-Yuan 33305, Taiwan, Republic of China d
Department of Neurosurgery, Chang Gung Memorial Hospital, 5 Fusing St., KweiShan, Tao-Yuan 33305, Taiwan, Republic of China
e
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, 155, Sec.2, Linong Street, Taipei 11221, Taiwan, Republic of China
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efficacy, because the target of the drug is usually located inside the tumor cells. In contrast, it is anticipated that less MNP uptake by vascular cells is preferred in targeted thrombolysis in order to preserve as much of the drug as possible in the lumen of the vessels. How the carrier system interacts with different cells may determine not only the efficacy of the therapeutic agents, but also their potential side effects. Nanoparticle uptake by cells is considered to be mediated predominantly via endocytosis,6–8 which may be inuenced by several parameters including the size and surface characteristics of nanoparticles.6–10 Based on the size of the particulate matter, endocytosis may be divided into two categories, phagocytosis and pinocytosis.6,7 Large particles at the micrometer scale may be engulfed into cells via phagocytosis.8 Uptake of particles with a smaller size range may be mediated by pinocytosis involving different molecules including clathrin or caveolin.7,8,11 Surface characteristics, such as charge, critically affect how nanoparticles interact with cells and proteins encountered in circulation.7,9 Because of the negatively charged property of cell membranes, it is anticipated that nanoparticles with a positively charged surface tend to interact with the plasma membrane more readily, causing more efficient internalization.6,9 It was also reported that nanoparticles with hydrophobic surface properties may have a higher cellular
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uptake than their counterparts with a hydrophilic surface, probably because of a stronger partition between the lipid-rich areas of the plasma membrane.12,13 In addition, the hydrophobicity of nanoparticles also affects protein adsorption, which is considered the key factor for opsonization, and consequently phagocytosis by the reticuloendothelial system,9,14 causing fast clearance in vivo.15 Coating drug carriers with poly(ethylene glycol) (PEG)3,16 has thus become a valid strategy to increase surface hydrophilicity, attenuate protein adsorption, potentially extend the duration in the bloodstream and, consequently, increase the targeting effects. Magnetic targeting may serve as an active strategy for targeted delivery of bioactive substances. It has been demonstrated that magnetic guiding may increase local retention of MNP–drug composites in vivo and increase the efficacy of glioma treatment.2,17 In magnetofection, the application of magnetic force to cultured cells may accelerate cellular uptake of MNPs, improve uptake of nucleic acids that are conjugated to MNPs, and thus increase the transfection efficiency.18,19 The enhancement effects may be due to magnetic eld-induced sedimentation,18,20 and increase both aggregation and adsorption of MNPs to the cell membrane.21 In addition, modication of the magnetic carriers with transfection reagents, such as poly(ethylene imine),22 may further improve their interaction with the plasma membrane, and subsequently improve transfection efficiency. Such modications may greatly enhance the efficacy of MNP uptake under magnetic inuence, and are thus, required for a satisfactory outcome. With a variety of carrier systems being tested in drug delivery, it is well known that the carrier system may interact with plasma proteins or chemical substances in biouids.23 It is anticipated that drug composites may encounter food components in biouids as well, but to our knowledge, no food component has been reported to interact with carrier systems and exerts an effect on cellular uptake or distribution of the carriers. Tea is a widely consumed beverage with great potential in the reduction of the incidence of many diseases,24–28 primarily due to its catechin components, including ()-epigallocatechin gallate (EGCG), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG), and ()-epicatechin (EC). Among these, EGCG is the most studied constituent of tea extract, and has been demonstrated to exert antioxidant,25,29 anti-angiogenesis,26,27,30 anti-proliferation,24,26 pro-apoptosis,24,26,27,30 and cancer-prevention effects.25,26 EGCG has been demonstrated to bind to receptors or proteins on the plasma membrane of tumor cells, such as the 67 kDa laminin receptor,24,26,31,32 resulting in inhibition of tumor growth and metastasis. In addition, EGCG may compete with or block the ligand/substrate binding for the epidermal growth factor receptor,24,30,33 and subsequently inhibit tumor growth. Therefore, catechins31 or related compounds34 have been formulated into targeting composites and demonstrated as potential anti-cancer therapeutics in cultured colorectal and breast tumor cells34 or in mice carrying prostate tumors.31 In this study, we tested a hypothesis that EGCG may exert an effect to enhance cellular uptake of MNPs by augmenting MNP–cell interactions; such effects may be further potentiated by application of magnetic force. Since PEG
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modication changes the surface characteristics of nanoparticles, and because PEG has been widely used in drug delivery systems to limit their elimination by the reticuloendothelial system,35 MNPs with or without PEG modication were compared in the study. The results suggested that EGCG may modify the surface characteristics of MNPs by adsorption, and thus enhance the interaction of MNPs and the plasma membrane of glioma cells.
Experimental section 1. Materials Magnetic nanoparticles, nanomag®-D COOH (MNP; 250 nm) and nanomag®-D PEG-COOH (MNP–PEG; 250 nm), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Fluorescent nano-screenMAG-CMX (CMX– MNP) was purchased from Chemicell GmbH (Berlin, Germany). Dulbecco's modied Eagle's medium (DMEM) with or without Nutrient Mixture F-12, M199 medium, and trypsin-ethylenediaminetetraacetic acid were purchased from Gibco BRL (Grand Island, NY, USA). Penicillin/streptomycin/amphotericin and endothelial cell growth supplement (ECGS) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Fetal bovine serum (FBS), heparin (50 000 units), ammonium persulfate, potassium thiocyanate (KSCN), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trimethylamine–diphenylhexatriene (TMA–DPH), benzyl alcohol, paraformaldehyde, EGCG, EGC, EC, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Burlingame, CA, USA). Rhodamine phalloidin, LysoTracker®, and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). Collagen (rat tail) was purchased from Roche (Indianapolis, IN, USA). An NdFeB magnet was purchased from New Favor Industry Co. (Taiwan). 2. Characterization of nanoparticles The hydrodynamic size distribution of MNPs was determined using a dynamic light scattering-based sub-micron particle size analyzer (N4 plus submicron particle size analyzer, Beckman Coulter, Inc., USA). The zeta potential of MNPs was determined using a particle size and zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd, Malvern, UK) in deionized water at 25 C. 3. Cell culture Glioma cells from human (LN-229) or rat (C6) were maintained in DMEM or DMEM/F-12 medium, respectively, containing 10% FBS and 1% penicillin/streptomycin/amphotericin mixture in a 37 C incubator supplied with 5% CO2. Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cords, the procedure being approved by the Institutional Review Board of Chang Gung Memorial Hospital. Briey, the lumen of the vein was washed with a cord buffer containing NaCl (137 mM), KCl (4 mM), HEPES (10 mM), and glucose (12 mM) at pH 7.4, followed by incubation of the cord with collagenase (0.1%) at 37 C in a 5% CO2 incubator for 8 min. Aer gentle mixing,
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the collagenase solution was collected and mixed with 10 ml of culture medium. The procedure was repeated 2–3 times. Aer centrifugation at 1400 rpm for 5 min, the cell pellet was suspended in 10 ml of culture medium containing M199 medium, FBS (10%), heparin (100 mg ml1), ECGS (30 mg ml1), and penicillin/streptomycin/amphotericin (1%) at pH 7.4. The cells were maintained at 37 C in an incubator supplied with 5% CO2 and used between passages 4 and 8. 4. Application of magnetic force A home-made magnetic plate was placed underneath a 24-well culture plate for application of magnetic dragging force. In this magnetic plate, 24 pieces of cylindrical NdFeB magnet with a diameter of 1.8 cm were arranged to provide a magnetic eld of 3.4 kG at the center of each well, as measured by a model 5180 Gauss meter (FW Bell, Orlando, FL, USA) with the probe placed at the surface of the magnet. In some experiments, the magnetic plate was placed underneath the culture plates for 5 min aer MNP administration, in order to facilitate sedimentation of the particles. 5. Confocal microscopy LN-229 cells were seeded onto coverslips 8 h before the experiments. The cells were incubated with green uorescent MNPs with carboxylmethyldextran coating (CMX–MNP; 100 mg ml1) and EGCG (6 mM) for 2 h. Aer addition of MNPs, the homemade magnetic plate was placed underneath the 24-well culture plate for 5 min followed by 2 h incubation at 37 C. Cells were then washed twice with ice-cold phosphate buffered saline (PBS), and xed with 4% paraformaldehyde. In some experiments, while cells were co-incubated with MNPs, LysoTracker® (0.125 mM) was used according to the manufacturer's instructions for staining of lysosomes. In other experiments, the cells were xed, permeabilized with Triton X-100 (0.5%; 10 min), and blocked with bovine serum albumin (0.5%; 1 h) prior to incubation with rhodamine phalloidin (1 : 60 dilution; 2 h) to staining the F-actin. The preparations were counterstained with DAPI and imaged with a LSM 510 Meta laser confocal microscope system (Carl Zeiss, Germany) tted with a 100/1.4 oil immersion objective lens. The quantitative analysis of colocalization was conducted using ImageJ soware. The Mander's overlap coefficient (R) was used for analysis of co-localization of MNP and lysosomes.36 6. Flow cytometry CMX–MNP (100 mg ml1) and EGCG (1 or 3 mM) were administered with the application of the magnet underneath the culture plates for 5 min to facilitate sedimentation. Aer incubation for 2 h, the cells were washed twice with ice-cold PBS before trypsinization prior to analysis. Aer centrifugation, the resulting cell pellet was resuspended in PBS, and subjected to analysis using a FACSCalibur ow cytometer (Becton Dickenson, Mississauga, CA, USA) with a 488 mm laser to determine uorescent signaling (15 000 cells per sample). The side scatter (SSC) distribution was gated at 400 based on normal cell conditions acquired from a control group without MNP or
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EGCG; the uorescence intensity was gated at 157 based on the distribution pattern acquired from the group without EGCG. 7. Cellular uptake determined by KSCN assay Cells were cultured in a 24-well culture plate until 80–90% conuence was achieved, before being exposed to MNP (100 mg ml1) and EGCG (0 to 20 mM) in the absence and presence of the NdFeB magnet for 24 h. In some experiments, cells were equilibrated at 4 C for 20 min, followed by an additional 1 h incubation with MNPs at 4 C in the absence and presence of the magnet. Aer trypsinization, the cell pellet was treated with 10% hydrochloric acid at 55 C for 4 h, followed by ammonium persulfate (1 mg ml1) and KSCN (1 M). The amount of cellassociated iron was determined with a VICTOR3 Multilabel Plate Reader (PerkinElmer, Shelton, CT, USA) at OD490. For calibration, a standard curve with a known amount of MNPs was prepared under identical conditions. 8. Antioxidant activity assay A stable free radical reagent, DPPH in ethanol was used to determine the scavenging activity of EGCG and N-acetylcysteine (NAC). Freshly prepared EGCG or NAC was mixed with DPPH (0.1 mM) at room temperature for 20 min, followed by determination of OD517. The actual decrease of DPPH absorption induced by EGCG was calculated by subtracting that of the control indicating the free radical scavenging activity. To evaluate the stability of MNP–PEG–EGCG aer storage time, the antioxidant activity of EGCG was determined. Briey, the supernatant solution and MNP–PEG–EGCG were magnetically separated followed by determination of the antioxidant activity of supernatant solution vs. particle suspension by the DPPH assay. 9. Plasma membrane uidity measurement To evaluate membrane uidity by uorescence spectroscopy, a C6 cell suspension (2.5 105 cells per ml) was treated with EGCG (10 mM) or benzyl alcohol (30 mM) in the presence of uorescence probe TMA–DPH (1 mM) at room temperature for 15 min. The light emission intensity of samples was measured using a Chameleon plate reader (Hidex, Mustionkatu, Finland). The excitation and emission wavelengths for TMA–DPH were 360 nm and 430 nm, respectively, and the uorescence anisotropy was calculated as described previously.37 The uorescence anisotropy values are inversely proportional to cell membrane uidity; a high degree of uorescence anisotropy represents a high structural order or a low cell membrane uidity. 10.
Preparation of MNP–PEG–EGCG or MNP–EGCG
EGCG (0.5 mg ml1) was mixed with MNP or MNP–PEG (5 mg ml1) in PBS and incubated at 37 C for 2 h. The composites were removed from un-adsorbed EGCG in solution by magnetic separation, suspended in PBS, and stored at 4 C. The amount of EGCG adsorbed on MNP–PEG or MNP was determined using a UV/visible spectrophotometer at OD280.
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11.
Cell toxicity assay
LN-229 cells were cultured in a 24-well plate to 80–90% conuence before incubation with MNPs (100 mg ml1) with free or conjugated EGCG (9.5 or 20 mM). Aer administration of MNP–EGCG, a magnet was placed underneath for 5 min or 2 h. Then, cells were washed with PBS and incubated with MTT (0.5 mg ml1) for an additional 1 h. The dark blue formazan crystals generated by the mitochondrial dehydrogenase were dissolved with dimethyl sulfoxide followed by measurement of OD540. 12.
Statistical analysis
Results are expressed as mean SE. Statistical evaluation of the data was performed with Student's t-test for simple comparisons between two values when appropriate. For multiple comparisons, results were analyzed by ANOVA followed by Duncan's post hoc test. A value of P < 0.05 was considered statistically signicant.
Results To observe the effects of EGCG on cellular uptake of MNPs, MNPs conjugated with a green uorophore (CMX–MNP) were incubated with LN-229 cells in the presence of EGCG (6 mM) for 2 h. Fig. 1 illustrates the related distribution of MNPs and lysosomes. Treatment with EGCG (Fig. 1B) increased the amount of MNPs in the cytoplasm compared with the PBS vehicle (Fig. 1A). The nucleus stained by DAPI was surrounded by green uorescence, suggesting that the MNPs were internalized in the cytoplasm without entering the nucleus. In
Fig. 1 Representative fluorescence images of cell-associated MNPs in response to EGCG treatment. LN-229 cells were incubated with CMX– MNP (100 mg ml1; green) in the presence of vehicle (PBS; A) or EGCG (6 mM; B) for 2 h, followed by counterstaining with LysoTracker (red) and DAPI (blue) before confocal microscopy.
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addition, the internalized MNPs were mostly co-localized with lysosomes in the peripheral region of the cell (Fig. 1B), suggesting involvement of endocytosis pathways. The amounts of MNPs co-localized with lysosomes were approximately 4-fold in response to EGCG treatment vs. PBS vehicle. The Mander's overlap coefficient (R) increased from 0.365 to 0.823 in response to EGCG treatment, suggesting co-localization of MNPs and lysosomes, and thus intracellular location of the MNPs. Cellassociated MNPs (MNPcell) measured by a KSCN assay revealed that EGCG (6 mM) signicantly increased cellular uptake of CMX–MNP from 3.5 0.1 to 20.9 0.1 mg per well (n ¼ 4, p < 0.05). Fig. 2 illustrates ow cytometry analysis of MNP uptake in response to EGCG. The relationship of forward scatter (FSC) vs. SSC is indicative of cell volume vs. complexity, respectively. The representative results illustrate that EGCG induced an increase in cell complexity at both 1 and 3 mM without alteration in cell volume (upper panel). The percentage of counts above the SSC value of 400 shied to the right in a concentration-dependent manner (middle panel), and averaged 22.9 8.9%, 41.2 5.0% and 61.4 2.7% in response to 0, 1 and 3 mM of EGCG, respectively (n ¼ 4). Similar results were observed with uorescence intensity above 157 (lower panel), suggesting an increase in cellular uptake of MNPs. The cell-associated uorescence intensity increased with EGCG concentration and averaged 50.4 15.7%, 64.9 12.6% and 78.5 8.1% in response to 0, 1 and 3 mM of EGCG, respectively (n ¼ 4). In
Fig. 2 Flow cytometry demonstration of a concentration-dependent enhancement effect of EGCG on MNP uptake. LN-229 cells were incubated with fluorescent CMX–MNP (100 mg ml1) and EGCG (1 or 3 mM) for 2 h prior to further analysis. The top panel illustrates the relationship between the cell size (forward scatter, FSC) and intracellular complexity (side scatter, SSC); the middle and bottom panels illustrate the distribution of intracellular complexity and fluorescence intensity, respectively. Effective cell counts in the R1 areas in the top panel were considered. The results are representative of four experiments.
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addition, cell viability was not signicantly altered aer incubation with MNPs (100 mg ml1) and EGCG (20 mM) for 24 h (n ¼ 4; data not shown). To determine the effect of EGCG and its interaction with magnetic force on MNP uptake, cultured LN-229 cells were incubated with MNPs and EGCG for 24 h prior to analysis by the KSCN assay. Fig. 3A illustrates that EGCG induced a concentration-dependent increase in cellular uptake of MNPs for all groups studied. In the absence of a magnetic eld, EGCG at 3 and 20 mM increased MNP uptake by 7- and 20-fold, respectively (n ¼ 4; P < 0.05). Similar results were observed with PEGmodied MNPs (MNP–PEG), however, modication with PEG caused a minor reduction of MNP uptake for most conditions. Magnets placed underneath the culture plates of LN-229 cells signicantly enhanced cellular uptake of MNPs with or without PEG by 1.5- to 2.8-fold. Because carrier-drug conjugates are usually administered via intravascular injection, whether EGCG exerts a similar effect on vascular endothelium may determine the targeting efficiency of the system. Thus, the effects of EGCG on HUVEC uptake of MNPs were examined. In contrast to an enhanced MNP uptake by glioma cells, EGCG exerted no effect on MNP uptake by HUVEC in the absence of a magnetic eld, however, in the presence of a magnet, EGCG at concentrations as low as 3 and 6 mM signicantly enhanced HUVEC uptake of MNPs and MNP– PEG, respectively (Fig. 3B), suggesting a synergetic effect of EGCG and magnetic eld on uptake by endothelial cells. In addition to EGCG, other catechins may exert similar effects on MNP uptake. The results demonstrated that EGC
Nanoscale
(Fig. 3C) and EC (Fig. 3D) also enhanced MNP uptake in a concentration-dependent manner with or without the magnet. Without the magnet, EGC at 6 mM induced a 2-fold increase in uptake of MNPs, but not in MNP–PEG; whereas with the magnet, uptake of MNPs and MNP–PEG increased by 2.3- and 2.0-fold in response to EGC, respectively. Similar enhancing effects on MNP uptake were also observed in response to EC. To determine whether the effects of EGCG are reversible, EGCG was removed from the culture medium prior to addition of MNPs to LN-229 cells (Fig. 4A). EGCG enhanced MNPcell for all conditions studied when EGCG was co-incubated with MNPs. In response to EGCG, MNPcell increased 5.4- and 21.9fold in the presence of the magnet for 5 min and 2 h, respectively. No signicant enhancement of MNP uptake was observed when cells were pre-exposed to EGCG followed by 2 h incubation with MNPs in the absence of EGCG, suggesting that the enhancement effect of EGCG requires the interaction of EGCG and MNPs. In addition, prolonged incubation with EGCG from
Fig. 4 The enhancing effect of EGCG on MNP uptake was transient
Fig. 3 Catechin-enhanced cellular uptake of MNPs. EGCG (A and B), epigallocatechin (EGC; C) and epicatechin (EC; D) induced a concentration-dependent effect on uptake of nanoparticles (MNPcell) by LN-229 cells (A, C, D) and HUVEC (B) in the presence or absence of the magnet (Mag). Cells were incubated with MNPs (100 mg ml1) with (+) or without () PEG coating for 24 h. Data are presented as mean SE (n ¼ 4). *: P < 0.05 compared with corresponding values without EGCG; †: P < 0.05 compared with corresponding values without magnetic field; #: P < 0.05 compared with the corresponding group without PEG modification.
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and reversible, but not mimicked by NAC. (A) LN-229 cells were incubated with MNPs (100 mg ml1; shaded area) with the magnet (Mag) underneath for 5 min or 2 h after addition of MNPs. Doubleheaded arrows indicate duration of EGCG treatment in groups (1)–(4). (B) LN-229 cells were incubated with MNPs (100 mg ml1) and NAC in the presence (+) or absence () of the magnet for 24 h. The antioxidant activity of EGCG and NAC was determined by the DPPH method (inset). Data are presented as mean SE (n ¼ 4–8). *: P < 0.05 compared with corresponding values without EGCG or NAC; †: P < 0.05 compared with corresponding values with Mag (5 min) or Mag (); §: P < 0.05 compared with the corresponding group with EGCG pre-treatment.
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2 to 6 h did not further enhance MNPcell. Similar effects were also observed for C6 cells (data not shown). NAC, with an antioxidant property similar to that of EGCG, did not exert an enhancement effect on MNP uptake (Fig. 4B), suggesting that the enhancement effect of EGCG may not be contributed to by its antioxidant property. To determine whether the enhancement effect of EGCG on MNP internalization may be predominantly mediated by endocytosis, experiments were conducted at 4 C. The application of magnetic force for 1 h signicantly enhanced MNPcell at 4 C, but not at 37 C. EGCG enhanced MNPcell by 1.6- and 3.6fold at 37 C in the presence of the magnet for 5 min and 1 h, respectively. At 4 C, the enhancement effect of EGCG was attenuated by 51% with the application of the magnet for 1 h, but not with the application of the magnet for 5 min (Fig. 5A). With confocal microscopy, internalization of MNPs was observed at 37 C, which was greatly enhanced by EGCG. Although MNPcell was not reduced by low temperature with 5 min of magnetic inuence (Fig. 5A), MNP internalization was not evident at 4 C (Fig. 5B). Quantitative results in response to EGCG treatment in Fig. 5A are modelled in Fig. 5C. In addition, EGCG may affect cell membrane uidity,35,36 which mediates the enhancement effects on MNP internalization. However, the concentration of EGCG used in the current study did not exert any effect on membrane uidity, as determined by membrane anisotropy (Fig. 5A, inset). Nevertheless, benzyl alcohol, an agent known to increase membrane uidity, decreased anisotropy but exerted no effect on cellular uptake of MNPs for all conditions studied (Fig. 5A), suggesting that the enhancement effect of EGCG may not be mediated by alteration of membrane uidity. To determine whether the enhancement effect of EGCG may be associated with modication of the surface characteristics of MNPs, effects of EGCG adsorption on MNPcell were examined. EGCG adsorption was performed by incubation of EGCG (1.1 mM) and MNP–PEG (5 mg ml1), followed by magnetic separation. Table 1 shows the size and zeta potential of EGCG-adsorbed vs. pristine MNPs. The adsorption of EGCG did not signicantly alter the size of the MNPs, however, EGCG adsorption altered the zeta potential of MNPs and MNP–PEG to approximately 30 mV. To determine the stability of MNP– EGCG composite, the antioxidant activity of EGCG associated with MNPs was determined. Aer magnetic separation, the antioxidant activity of EGCG was primarily associated with the composite aer storage at 4 C for 1 to 24 h; the antioxidant activity in the supernatant solution was only 2.4–5.5% of that associated with the particles aer magnetic separation. These results demonstrated that adsorbed EGCG remained stable with the MNPs during the 24 h period (Fig. 6A). In all groups studied with LN-229 cells, application of a magnetic eld for 24 h signicantly enhanced MNP uptake by 1.4- to 9.6-fold (P < 0.05; Fig. 6B). With the same amount of EGCG of 6.4 mM, MNP uptake enhancements induced by MNP-associated EGCG (bound) were 10.3 and 2.5 times that induced by free EGCG in the absence and presence of the magnet, respectively (P < 0.05). With EGCG concentration of 12.8 mM, bound EGCG-induced enhancing effect on MNP uptake was 8.1 and 1.1 times that induced by free
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Fig. 5 EGCG increased cell-associated MNPs at 4 C and 37 C without altering the fluidity of plasma membrane. C6 (A) or LN-229 (B) cells were incubated at 4 vs. 37 C for 20 min before administration of MNPs (A) or CMX–MNP (B) with EGCG (10 mM) or benzyl alcohol (15– 30 mM), followed by additional 1 h (A and B) or 15 min (A, inset) incubation. After the treatment, the cells were counterstained with rhodamine phalloidin (red) and DAPI (blue) prior to confocal microscopy (B). The schematic diagrams (C) illustrate the relative amount of cell-associated MNPs (MNPcell) measured in (A). Values are presented as mean SE (n ¼ 4–8). *: P < 0.05 compared with corresponding vehicle group; #: P < 0.05 compared with corresponding group at 4 C; §: P < 0.05 compared with the corresponding group with 5 min magnetic field.
EGCG in the absence and presence of the magnet, respectively (P < 0.05). Similar results were also observed in culture of HUVEC in response to EGCG (Fig. 6C). Adsorption of EGCG enhanced MNP uptake by 6.7- and 8.3-fold in the absence and presence of the magnet, respectively; adsorption of EGCG
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Table 1
Nanoscale Size and zeta potential of MNPsa
Particle
Size (nm)
Zeta potential (mV)
MNP MNP-PEG MNP-EGCG MNP-PEG-EGCG
263.7 5.5 299.8 3.6 270.3 6.3 296.3 4.0
40.1 11.3 25.5 31.9
a
0.3 0.4 0.1 0.5
Data presented as mean SE, n ¼ 3.
enhanced MNP–PEG uptake by 7- and 20-fold in the absence and presence of the magnet, respectively. These ndings suggest that EGCG adsorption may account for the enhanced uptake of MNPs. The results of the MTT assay demonstrated that there was no signicant cytotoxicity in the groups studied except for MNP-EGCG (Fig. 7), which was probably because of a higher concentration of EGCG in the cell surroundings with the MNP–EGCG group in the presence of the magnet.
Discussion Nanoparticles have been tested as drug carriers in therapeutics.1,14,38 Interaction of the carriers with a biological system may determine the efficacy of the pharmacological treatment. In this study, we have demonstrated a novel effect of food components: tea catechins, on cellular uptake of nanoparticles. The enhancement effects of EGCG on cellular uptake of MNPs appear to be cell-specic, concentration-dependent, and synergized with magnetic force application. Our study also reveals a potential food–drug interaction that may have an impact on pharmacokinetics and toxicity of nanocarriers. The results demonstrate that EGCG signicantly increased MNPs in cytoplasm, which were co-localized with lysosomes as observed using confocal microscopy, suggesting that MNPs were internalized into cytoplasm via endocytosis. The imaging results were consistent with those from ow cytometry and KSCN quantication of iron, and were in agreement with those reported in the literature7–9,39–42 demonstrating that the main route of nanoparticles entering cells was via endocytosis, both in glioma39,42 as well as in normal cells.40,41 Although the colorimetric quantication of iron may not allow differentiation of MNPs in the cytoplasm from those on the plasma membrane,21 the assay provides quantitative determination of cell-associated MNPs (MNPcell), which reveal the consequence of particle–plasma membrane interaction. Whether EGCG exerts any effect on particles escaping from lysosomes or exocytosis remains to be determined. Our results demonstrate that EGCG exerted a signicant enhancement effect on MNPcell even at 4 C (Fig. 5A) when energy-dependent endocytosis was blocked.43 Thus, the effect of EGCG may be mediated by, at least in part, a mechanism other than endocytosis. Similar effects were observed aer application of magnetic force at 4 C for one hour, which is consistent with a recent observation that magnetic eld may increase MNP attachment to the cells at 4 C.44 It is likely that EGCG may increase MNPcell by enhancing the adsorption of MNPs to the
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Adsorption of EGCG enhanced MNP uptake by glioma and endothelial cells. The MNP–PEG–EGCG complex was produced by incubation of EGCG and MNPs or MNP–PEG at 37 C for 2 h. The stability of the complex at 4 C was determined by measuring the antioxidant activity of EGCG associated with the particles (A). The effects of free (f) vs. bound (b) forms of EGCG (6.4 and 12.8 mM) with MNPs (100 mg ml1) in LN-229 cells (B) and the effects of EGCG (10 mM) adsorption on uptake of MNPs or MNP–PEG (140 mg ml1) by HUVEC (C) for 24 h were determined. Values are presented as mean SE. §: P < 0.05 compared with corresponding MNP–PEG–EGCG values; *: P < 0.05 compared with corresponding groups without magnetic field; †: P < 0.05 compared with the corresponding free form groups; #: P < 0.05 compared with the corresponding group with lower EGCG concentration or without EGCG. Fig. 6
plasma membrane of glioma cells. In addition, magnetic force may further increase MNP attachment to the plasma membrane in the presence of EGCG, as demonstrated by a synergistic effect of magnetic force and EGCG (Fig. 5C). However, endocytosis may play a pivotal role in the synergistic effects of EGCG and magnetic force, because the value of MNPcell doubled at 37 C vs. 4 C in the presence of both EGCG
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Fig. 7 Cytotoxicity of MNPs/MNP–PEG with free or bound EGCG under the influence of magnetic field for 5 min vs. 2 h. LN229 cells were incubated with MNPs or MNP–PEG (100 mg ml1) in the presence of free (f) or bound (b) form of EGCG (9.5 mM) for 2 h. Values are presented as mean SE (n ¼ 6). *, P < 0.05 compared with the control group without nanoparticles or EGCG; †, P < 0.05 compared with the corresponding groups with 5 min magnetic field.
and the magnet. Thus, the enhancement effect of EGCG at 37 C may be mediated by increasing both membrane-bound and internalized MNPs (Fig. 5C). The interaction of MNPs and plasma membrane appears to be strong enough to be retained aer the PBS wash, if not internalized. Previous studies have demonstrated that nanoparticles may induce membrane perturbation.45,46 Such membrane ruffling may provide a physical barrier during the wash procedure and be responsible, at least in part, for an increased MNPcell in response to EGCG or magnetic force. When endocytosis was blocked by low temperature, the enhancement effect of EGCG may be because of chemical interaction of MNPs and plasma membrane, suggesting that EGCG may modify the surface characteristics of MNPs. In addition, the enhancing effect of EGCG was not observed when EGCG was removed during administration of MNPs to the culture (Fig. 4A), suggesting that the co-existence of EGCG and MNPs appears to be required for EGCG to have its enhancing effects. Although EGCG may bind to proteins on the plasma membrane and activate certain signaling pathways,24,26 the enhancing effects on MNP uptake appear to be acute and reversible, which are independent of signaling pathways that require protein phosphorylation or gene expression. It is likely that the enhancing effect of EGCG might be a result of a direct interaction of EGCG with plasma membrane of tumor cells. It has been reported that nanoparticles with a hydrophobic surface may interact more readily with the lipid bilayer of the plasma membrane.12,13,47 EGCG exerts a calculated octanol–water partition coefficient (log P) of 2.29, suggesting that it has hydrophobic characteristics. Previous studies have also reported that EGCG and other catechins may form hydrogen bonds with lipid bilayers.48,49 Alternatively, EGCG may bind to membrane receptors, including a 67 kDa laminin receptor,24,31,32 or specic membrane regions such as lipid ras.24,50 All the above mentioned mechanisms of EGCG–membrane interaction may contribute to the enhancing effects of EGCG on MNP uptake. In addition, the zeta potential of MNPs is one of many factors that may affect how MNPs interact with cells. The zeta potential
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changes to the range of 25 to 32 mV aer EGCG adsorption in distilled water, demonstrating the effect of EGCG on the surface characteristics of the MNPs. Nevertheless, previous studies have shown that the zeta potential of nanoparticles was between 9 and 19 mV aer incubation in serum-supplemented culture medium despite the original zeta potential,51,52 which is probably due to the masking effect of serum proteins in the medium. Therefore, the enhancing effect of EGCG on MNP uptake is unlikely to be the result of the contribution of changes of zeta potential in the culture medium. The enhancing effect on MNP cellular uptake was also observed aer EGCG modication of MNPs via adsorption and magnetic separation. With the same concentration of EGCG in culture, greater MNPcell was observed with the bound vs. free form of EGCG (Fig. 6B), which was probably because of more EGCG coming into contact with the plasma membrane of the cell following MNP sedimentation. In spite of poor bioavailability of tea catechins,26,53 oral consumption of tea preparations may achieve a peak plasma concentration of 5–7 mM,26 which is within the range of concentrations for EGCG that exerted an enhancing effect in the current study. As shown in Fig. 6B, EGCG-bound MNPs had a signicantly higher MNPcell. It is thus anticipated that MNP–catechin interactions may allow MNP retention at cell surroundings with local concentration of catechins much higher than that in plasma. However, whether the enhanced MNP–plasma membrane interaction may occur in tumors remains to be determined. The effect of EGCG was cell-specic, because no effect was observed on MNP uptake by HUVEC without a magnetic eld. Nevertheless, the magnetic force signicantly enhanced MNP uptake by HUVEC, especially for MNPs not modied by PEG. The underlying mechanism that causes cell specicity is uncertain. Whether the difference may be because of characteristics of the plasma membrane of normal vs. tumor cells remains to be determined. Nevertheless, PEG modication may jeopardize particle internalization by the target cells, but the uptake of PEG-modied MNPs may be restored by co-administration of EGCG and a magnetic eld. The effects of PEG on tumor cell uptake of nanoparticles are consistent with the previous ndings that lower transfection efficiency was associated with carriers modied by PEG.54 It has also been demonstrated that EGCG may be incorporated into a nanocarrier composite for treatment of prostate cancer in vitro55 and in animal models.31 Although EGCGinduced growth inhibition may play a role in the previously mentioned studies, it may be that EGCG can augment the therapeutic effects by enhancing nanoparticle uptake in these systems. The current studies may provide a novel interpretation of the effect of EGCG in the nanocarrier systems for treatment of cancer.
Conclusions We demonstrated a feasible application of a food component in nanocarrier systems for targeted delivery of drugs or biological molecules by MNPs. Combined application of EGCG and a magnetic eld may markedly increase MNP uptake, which may
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lead to better therapeutic or transfection efficacy. In addition, MNP–EGCG composites may be produced by physical adsorption, which is a simple approach to facilitate tumor uptake of MNPs while sustaining the bioactivity in circulation. With cell specicity, the targeting effects of the system may also be augmented, which allows extensive tumor uptake but spares the non-targets, such as vascular endothelial cells. Combined application of magnetic force and EGCG may facilitate particle internalization in targeted therapeutics or magnetofection.
Acknowledgements We thank the Ministry of Science and Technology of the Republic of China (NSC 100-2120-M-182-001- and NSC 1012320-B-182-038-), Healthy Aging Research Program at Chang Gung University (EMRPD1A0841), and Chang-Gung Memorial Hospital (CMRPD1D0231) for funding support. The authors thank Dr Ying-Tung Lau at Chang Gung University of Science and Technology for assistance in HUVEC culture, and Dr HawMing Huang at Taipei Medical University for assistance in uorescence anisotropy measurement.
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Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle–cell interaction Yi-Ching Lu,ae Pei-Chun Luo,a Chun-Wan Huang,a Yann-Lii Leu,b Tzu-Hao Wang,c Kuo-Chen Wei,d Hsin-Ell Wange and Yunn-Hwa Ma*a Nanoparticles may serve as carriers in targeted therapeutics; interaction of the nanoparticles with a biological system may determine their targeting effects and therapeutic efficacy. Epigallocatechin-3gallate (EGCG), a major component of tea catechins, has been conjugated with nanoparticles and tested as an anticancer agent. We investigated whether EGCG may enhance nanoparticle uptake by tumor cells. Cellular uptake of a dextran-coated magnetic nanoparticle (MNP) was determined by confocal microscopy, flow cytometry or a potassium thiocyanate colorimetric method. We demonstrated that EGCG greatly enhanced interaction and/or internalization of MNPs (with or without polyethylene glycol) by glioma cells, but not vascular endothelial cells. The enhancing effects are both time- and concentration-dependent. Such effects may be induced by a simple mix of MNPs with EGCG at a concentration as low as 1–3 mM, which increased MNP uptake 2- to 7-fold. In addition, application of magnetic force further potentiated MNP uptake, suggesting a synergetic effect of EGCG and magnetic force. Because the effects of EGCG were preserved at 4 C, but not when EGCG was removed from the
Received 2nd February 2014 Accepted 24th June 2014
culture medium prior to addition of MNPs, a direct interaction of EGCG and MNPs was implicated. Use of an MNP–EGCG composite produced by adsorption of EGCG and magnetic separation also led to an
DOI: 10.1039/c4nr00617h
enhanced uptake. The results reveal a novel interaction of a food component and nanocarrier system,
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which may be potentially amenable to magnetofection, cell labeling/tracing, and targeted therapeutics.
Introduction In targeted delivery of therapeutic agents, a targeting carrier is expected to increase drug concentration at the target site and thus reduce systemic distribution/side effects. A magnetic nanoparticle (MNP), composed of an iron oxide core and a polymer coating, has been tested in animal models as a magnetically guided carrier in the delivery of chemotherapeutic1–3 or thrombolytic agents.4,5 In such biomedical applications, the interaction of cells and nanoparticles is crucial for the pharmacological efficacy of the carrier-drug system, depending on the sites of the target. In targeted chemotherapeutics, MNP internalization by tumor cells may determine the therapeutic a
Department of Physiology and Pharmacology, Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, Republic of China. E-mail: [email protected] b
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, Republic of China
c Genomic Medicine Research Core Laboratory, Chang Gung Memorial Hospital, 5 Fusing St., Kwei-Shan, Tao-Yuan 33305, Taiwan, Republic of China d
Department of Neurosurgery, Chang Gung Memorial Hospital, 5 Fusing St., KweiShan, Tao-Yuan 33305, Taiwan, Republic of China
e
Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, 155, Sec.2, Linong Street, Taipei 11221, Taiwan, Republic of China
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efficacy, because the target of the drug is usually located inside the tumor cells. In contrast, it is anticipated that less MNP uptake by vascular cells is preferred in targeted thrombolysis in order to preserve as much of the drug as possible in the lumen of the vessels. How the carrier system interacts with different cells may determine not only the efficacy of the therapeutic agents, but also their potential side effects. Nanoparticle uptake by cells is considered to be mediated predominantly via endocytosis,6–8 which may be inuenced by several parameters including the size and surface characteristics of nanoparticles.6–10 Based on the size of the particulate matter, endocytosis may be divided into two categories, phagocytosis and pinocytosis.6,7 Large particles at the micrometer scale may be engulfed into cells via phagocytosis.8 Uptake of particles with a smaller size range may be mediated by pinocytosis involving different molecules including clathrin or caveolin.7,8,11 Surface characteristics, such as charge, critically affect how nanoparticles interact with cells and proteins encountered in circulation.7,9 Because of the negatively charged property of cell membranes, it is anticipated that nanoparticles with a positively charged surface tend to interact with the plasma membrane more readily, causing more efficient internalization.6,9 It was also reported that nanoparticles with hydrophobic surface properties may have a higher cellular
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uptake than their counterparts with a hydrophilic surface, probably because of a stronger partition between the lipid-rich areas of the plasma membrane.12,13 In addition, the hydrophobicity of nanoparticles also affects protein adsorption, which is considered the key factor for opsonization, and consequently phagocytosis by the reticuloendothelial system,9,14 causing fast clearance in vivo.15 Coating drug carriers with poly(ethylene glycol) (PEG)3,16 has thus become a valid strategy to increase surface hydrophilicity, attenuate protein adsorption, potentially extend the duration in the bloodstream and, consequently, increase the targeting effects. Magnetic targeting may serve as an active strategy for targeted delivery of bioactive substances. It has been demonstrated that magnetic guiding may increase local retention of MNP–drug composites in vivo and increase the efficacy of glioma treatment.2,17 In magnetofection, the application of magnetic force to cultured cells may accelerate cellular uptake of MNPs, improve uptake of nucleic acids that are conjugated to MNPs, and thus increase the transfection efficiency.18,19 The enhancement effects may be due to magnetic eld-induced sedimentation,18,20 and increase both aggregation and adsorption of MNPs to the cell membrane.21 In addition, modication of the magnetic carriers with transfection reagents, such as poly(ethylene imine),22 may further improve their interaction with the plasma membrane, and subsequently improve transfection efficiency. Such modications may greatly enhance the efficacy of MNP uptake under magnetic inuence, and are thus, required for a satisfactory outcome. With a variety of carrier systems being tested in drug delivery, it is well known that the carrier system may interact with plasma proteins or chemical substances in biouids.23 It is anticipated that drug composites may encounter food components in biouids as well, but to our knowledge, no food component has been reported to interact with carrier systems and exerts an effect on cellular uptake or distribution of the carriers. Tea is a widely consumed beverage with great potential in the reduction of the incidence of many diseases,24–28 primarily due to its catechin components, including ()-epigallocatechin gallate (EGCG), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG), and ()-epicatechin (EC). Among these, EGCG is the most studied constituent of tea extract, and has been demonstrated to exert antioxidant,25,29 anti-angiogenesis,26,27,30 anti-proliferation,24,26 pro-apoptosis,24,26,27,30 and cancer-prevention effects.25,26 EGCG has been demonstrated to bind to receptors or proteins on the plasma membrane of tumor cells, such as the 67 kDa laminin receptor,24,26,31,32 resulting in inhibition of tumor growth and metastasis. In addition, EGCG may compete with or block the ligand/substrate binding for the epidermal growth factor receptor,24,30,33 and subsequently inhibit tumor growth. Therefore, catechins31 or related compounds34 have been formulated into targeting composites and demonstrated as potential anti-cancer therapeutics in cultured colorectal and breast tumor cells34 or in mice carrying prostate tumors.31 In this study, we tested a hypothesis that EGCG may exert an effect to enhance cellular uptake of MNPs by augmenting MNP–cell interactions; such effects may be further potentiated by application of magnetic force. Since PEG
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modication changes the surface characteristics of nanoparticles, and because PEG has been widely used in drug delivery systems to limit their elimination by the reticuloendothelial system,35 MNPs with or without PEG modication were compared in the study. The results suggested that EGCG may modify the surface characteristics of MNPs by adsorption, and thus enhance the interaction of MNPs and the plasma membrane of glioma cells.
Experimental section 1. Materials Magnetic nanoparticles, nanomag®-D COOH (MNP; 250 nm) and nanomag®-D PEG-COOH (MNP–PEG; 250 nm), were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). Fluorescent nano-screenMAG-CMX (CMX– MNP) was purchased from Chemicell GmbH (Berlin, Germany). Dulbecco's modied Eagle's medium (DMEM) with or without Nutrient Mixture F-12, M199 medium, and trypsin-ethylenediaminetetraacetic acid were purchased from Gibco BRL (Grand Island, NY, USA). Penicillin/streptomycin/amphotericin and endothelial cell growth supplement (ECGS) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Fetal bovine serum (FBS), heparin (50 000 units), ammonium persulfate, potassium thiocyanate (KSCN), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trimethylamine–diphenylhexatriene (TMA–DPH), benzyl alcohol, paraformaldehyde, EGCG, EGC, EC, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Burlingame, CA, USA). Rhodamine phalloidin, LysoTracker®, and 40 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Carlsbad, CA, USA). Collagen (rat tail) was purchased from Roche (Indianapolis, IN, USA). An NdFeB magnet was purchased from New Favor Industry Co. (Taiwan). 2. Characterization of nanoparticles The hydrodynamic size distribution of MNPs was determined using a dynamic light scattering-based sub-micron particle size analyzer (N4 plus submicron particle size analyzer, Beckman Coulter, Inc., USA). The zeta potential of MNPs was determined using a particle size and zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd, Malvern, UK) in deionized water at 25 C. 3. Cell culture Glioma cells from human (LN-229) or rat (C6) were maintained in DMEM or DMEM/F-12 medium, respectively, containing 10% FBS and 1% penicillin/streptomycin/amphotericin mixture in a 37 C incubator supplied with 5% CO2. Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cords, the procedure being approved by the Institutional Review Board of Chang Gung Memorial Hospital. Briey, the lumen of the vein was washed with a cord buffer containing NaCl (137 mM), KCl (4 mM), HEPES (10 mM), and glucose (12 mM) at pH 7.4, followed by incubation of the cord with collagenase (0.1%) at 37 C in a 5% CO2 incubator for 8 min. Aer gentle mixing,
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the collagenase solution was collected and mixed with 10 ml of culture medium. The procedure was repeated 2–3 times. Aer centrifugation at 1400 rpm for 5 min, the cell pellet was suspended in 10 ml of culture medium containing M199 medium, FBS (10%), heparin (100 mg ml1), ECGS (30 mg ml1), and penicillin/streptomycin/amphotericin (1%) at pH 7.4. The cells were maintained at 37 C in an incubator supplied with 5% CO2 and used between passages 4 and 8. 4. Application of magnetic force A home-made magnetic plate was placed underneath a 24-well culture plate for application of magnetic dragging force. In this magnetic plate, 24 pieces of cylindrical NdFeB magnet with a diameter of 1.8 cm were arranged to provide a magnetic eld of 3.4 kG at the center of each well, as measured by a model 5180 Gauss meter (FW Bell, Orlando, FL, USA) with the probe placed at the surface of the magnet. In some experiments, the magnetic plate was placed underneath the culture plates for 5 min aer MNP administration, in order to facilitate sedimentation of the particles. 5. Confocal microscopy LN-229 cells were seeded onto coverslips 8 h before the experiments. The cells were incubated with green uorescent MNPs with carboxylmethyldextran coating (CMX–MNP; 100 mg ml1) and EGCG (6 mM) for 2 h. Aer addition of MNPs, the homemade magnetic plate was placed underneath the 24-well culture plate for 5 min followed by 2 h incubation at 37 C. Cells were then washed twice with ice-cold phosphate buffered saline (PBS), and xed with 4% paraformaldehyde. In some experiments, while cells were co-incubated with MNPs, LysoTracker® (0.125 mM) was used according to the manufacturer's instructions for staining of lysosomes. In other experiments, the cells were xed, permeabilized with Triton X-100 (0.5%; 10 min), and blocked with bovine serum albumin (0.5%; 1 h) prior to incubation with rhodamine phalloidin (1 : 60 dilution; 2 h) to staining the F-actin. The preparations were counterstained with DAPI and imaged with a LSM 510 Meta laser confocal microscope system (Carl Zeiss, Germany) tted with a 100/1.4 oil immersion objective lens. The quantitative analysis of colocalization was conducted using ImageJ soware. The Mander's overlap coefficient (R) was used for analysis of co-localization of MNP and lysosomes.36 6. Flow cytometry CMX–MNP (100 mg ml1) and EGCG (1 or 3 mM) were administered with the application of the magnet underneath the culture plates for 5 min to facilitate sedimentation. Aer incubation for 2 h, the cells were washed twice with ice-cold PBS before trypsinization prior to analysis. Aer centrifugation, the resulting cell pellet was resuspended in PBS, and subjected to analysis using a FACSCalibur ow cytometer (Becton Dickenson, Mississauga, CA, USA) with a 488 mm laser to determine uorescent signaling (15 000 cells per sample). The side scatter (SSC) distribution was gated at 400 based on normal cell conditions acquired from a control group without MNP or
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Nanoscale
EGCG; the uorescence intensity was gated at 157 based on the distribution pattern acquired from the group without EGCG. 7. Cellular uptake determined by KSCN assay Cells were cultured in a 24-well culture plate until 80–90% conuence was achieved, before being exposed to MNP (100 mg ml1) and EGCG (0 to 20 mM) in the absence and presence of the NdFeB magnet for 24 h. In some experiments, cells were equilibrated at 4 C for 20 min, followed by an additional 1 h incubation with MNPs at 4 C in the absence and presence of the magnet. Aer trypsinization, the cell pellet was treated with 10% hydrochloric acid at 55 C for 4 h, followed by ammonium persulfate (1 mg ml1) and KSCN (1 M). The amount of cellassociated iron was determined with a VICTOR3 Multilabel Plate Reader (PerkinElmer, Shelton, CT, USA) at OD490. For calibration, a standard curve with a known amount of MNPs was prepared under identical conditions. 8. Antioxidant activity assay A stable free radical reagent, DPPH in ethanol was used to determine the scavenging activity of EGCG and N-acetylcysteine (NAC). Freshly prepared EGCG or NAC was mixed with DPPH (0.1 mM) at room temperature for 20 min, followed by determination of OD517. The actual decrease of DPPH absorption induced by EGCG was calculated by subtracting that of the control indicating the free radical scavenging activity. To evaluate the stability of MNP–PEG–EGCG aer storage time, the antioxidant activity of EGCG was determined. Briey, the supernatant solution and MNP–PEG–EGCG were magnetically separated followed by determination of the antioxidant activity of supernatant solution vs. particle suspension by the DPPH assay. 9. Plasma membrane uidity measurement To evaluate membrane uidity by uorescence spectroscopy, a C6 cell suspension (2.5 105 cells per ml) was treated with EGCG (10 mM) or benzyl alcohol (30 mM) in the presence of uorescence probe TMA–DPH (1 mM) at room temperature for 15 min. The light emission intensity of samples was measured using a Chameleon plate reader (Hidex, Mustionkatu, Finland). The excitation and emission wavelengths for TMA–DPH were 360 nm and 430 nm, respectively, and the uorescence anisotropy was calculated as described previously.37 The uorescence anisotropy values are inversely proportional to cell membrane uidity; a high degree of uorescence anisotropy represents a high structural order or a low cell membrane uidity. 10.
Preparation of MNP–PEG–EGCG or MNP–EGCG
EGCG (0.5 mg ml1) was mixed with MNP or MNP–PEG (5 mg ml1) in PBS and incubated at 37 C for 2 h. The composites were removed from un-adsorbed EGCG in solution by magnetic separation, suspended in PBS, and stored at 4 C. The amount of EGCG adsorbed on MNP–PEG or MNP was determined using a UV/visible spectrophotometer at OD280.
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11.
Cell toxicity assay
LN-229 cells were cultured in a 24-well plate to 80–90% conuence before incubation with MNPs (100 mg ml1) with free or conjugated EGCG (9.5 or 20 mM). Aer administration of MNP–EGCG, a magnet was placed underneath for 5 min or 2 h. Then, cells were washed with PBS and incubated with MTT (0.5 mg ml1) for an additional 1 h. The dark blue formazan crystals generated by the mitochondrial dehydrogenase were dissolved with dimethyl sulfoxide followed by measurement of OD540. 12.
Statistical analysis
Results are expressed as mean SE. Statistical evaluation of the data was performed with Student's t-test for simple comparisons between two values when appropriate. For multiple comparisons, results were analyzed by ANOVA followed by Duncan's post hoc test. A value of P < 0.05 was considered statistically signicant.
Results To observe the effects of EGCG on cellular uptake of MNPs, MNPs conjugated with a green uorophore (CMX–MNP) were incubated with LN-229 cells in the presence of EGCG (6 mM) for 2 h. Fig. 1 illustrates the related distribution of MNPs and lysosomes. Treatment with EGCG (Fig. 1B) increased the amount of MNPs in the cytoplasm compared with the PBS vehicle (Fig. 1A). The nucleus stained by DAPI was surrounded by green uorescence, suggesting that the MNPs were internalized in the cytoplasm without entering the nucleus. In
Fig. 1 Representative fluorescence images of cell-associated MNPs in response to EGCG treatment. LN-229 cells were incubated with CMX– MNP (100 mg ml1; green) in the presence of vehicle (PBS; A) or EGCG (6 mM; B) for 2 h, followed by counterstaining with LysoTracker (red) and DAPI (blue) before confocal microscopy.
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addition, the internalized MNPs were mostly co-localized with lysosomes in the peripheral region of the cell (Fig. 1B), suggesting involvement of endocytosis pathways. The amounts of MNPs co-localized with lysosomes were approximately 4-fold in response to EGCG treatment vs. PBS vehicle. The Mander's overlap coefficient (R) increased from 0.365 to 0.823 in response to EGCG treatment, suggesting co-localization of MNPs and lysosomes, and thus intracellular location of the MNPs. Cellassociated MNPs (MNPcell) measured by a KSCN assay revealed that EGCG (6 mM) signicantly increased cellular uptake of CMX–MNP from 3.5 0.1 to 20.9 0.1 mg per well (n ¼ 4, p < 0.05). Fig. 2 illustrates ow cytometry analysis of MNP uptake in response to EGCG. The relationship of forward scatter (FSC) vs. SSC is indicative of cell volume vs. complexity, respectively. The representative results illustrate that EGCG induced an increase in cell complexity at both 1 and 3 mM without alteration in cell volume (upper panel). The percentage of counts above the SSC value of 400 shied to the right in a concentration-dependent manner (middle panel), and averaged 22.9 8.9%, 41.2 5.0% and 61.4 2.7% in response to 0, 1 and 3 mM of EGCG, respectively (n ¼ 4). Similar results were observed with uorescence intensity above 157 (lower panel), suggesting an increase in cellular uptake of MNPs. The cell-associated uorescence intensity increased with EGCG concentration and averaged 50.4 15.7%, 64.9 12.6% and 78.5 8.1% in response to 0, 1 and 3 mM of EGCG, respectively (n ¼ 4). In
Fig. 2 Flow cytometry demonstration of a concentration-dependent enhancement effect of EGCG on MNP uptake. LN-229 cells were incubated with fluorescent CMX–MNP (100 mg ml1) and EGCG (1 or 3 mM) for 2 h prior to further analysis. The top panel illustrates the relationship between the cell size (forward scatter, FSC) and intracellular complexity (side scatter, SSC); the middle and bottom panels illustrate the distribution of intracellular complexity and fluorescence intensity, respectively. Effective cell counts in the R1 areas in the top panel were considered. The results are representative of four experiments.
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addition, cell viability was not signicantly altered aer incubation with MNPs (100 mg ml1) and EGCG (20 mM) for 24 h (n ¼ 4; data not shown). To determine the effect of EGCG and its interaction with magnetic force on MNP uptake, cultured LN-229 cells were incubated with MNPs and EGCG for 24 h prior to analysis by the KSCN assay. Fig. 3A illustrates that EGCG induced a concentration-dependent increase in cellular uptake of MNPs for all groups studied. In the absence of a magnetic eld, EGCG at 3 and 20 mM increased MNP uptake by 7- and 20-fold, respectively (n ¼ 4; P < 0.05). Similar results were observed with PEGmodied MNPs (MNP–PEG), however, modication with PEG caused a minor reduction of MNP uptake for most conditions. Magnets placed underneath the culture plates of LN-229 cells signicantly enhanced cellular uptake of MNPs with or without PEG by 1.5- to 2.8-fold. Because carrier-drug conjugates are usually administered via intravascular injection, whether EGCG exerts a similar effect on vascular endothelium may determine the targeting efficiency of the system. Thus, the effects of EGCG on HUVEC uptake of MNPs were examined. In contrast to an enhanced MNP uptake by glioma cells, EGCG exerted no effect on MNP uptake by HUVEC in the absence of a magnetic eld, however, in the presence of a magnet, EGCG at concentrations as low as 3 and 6 mM signicantly enhanced HUVEC uptake of MNPs and MNP– PEG, respectively (Fig. 3B), suggesting a synergetic effect of EGCG and magnetic eld on uptake by endothelial cells. In addition to EGCG, other catechins may exert similar effects on MNP uptake. The results demonstrated that EGC
Nanoscale
(Fig. 3C) and EC (Fig. 3D) also enhanced MNP uptake in a concentration-dependent manner with or without the magnet. Without the magnet, EGC at 6 mM induced a 2-fold increase in uptake of MNPs, but not in MNP–PEG; whereas with the magnet, uptake of MNPs and MNP–PEG increased by 2.3- and 2.0-fold in response to EGC, respectively. Similar enhancing effects on MNP uptake were also observed in response to EC. To determine whether the effects of EGCG are reversible, EGCG was removed from the culture medium prior to addition of MNPs to LN-229 cells (Fig. 4A). EGCG enhanced MNPcell for all conditions studied when EGCG was co-incubated with MNPs. In response to EGCG, MNPcell increased 5.4- and 21.9fold in the presence of the magnet for 5 min and 2 h, respectively. No signicant enhancement of MNP uptake was observed when cells were pre-exposed to EGCG followed by 2 h incubation with MNPs in the absence of EGCG, suggesting that the enhancement effect of EGCG requires the interaction of EGCG and MNPs. In addition, prolonged incubation with EGCG from
Fig. 4 The enhancing effect of EGCG on MNP uptake was transient
Fig. 3 Catechin-enhanced cellular uptake of MNPs. EGCG (A and B), epigallocatechin (EGC; C) and epicatechin (EC; D) induced a concentration-dependent effect on uptake of nanoparticles (MNPcell) by LN-229 cells (A, C, D) and HUVEC (B) in the presence or absence of the magnet (Mag). Cells were incubated with MNPs (100 mg ml1) with (+) or without () PEG coating for 24 h. Data are presented as mean SE (n ¼ 4). *: P < 0.05 compared with corresponding values without EGCG; †: P < 0.05 compared with corresponding values without magnetic field; #: P < 0.05 compared with the corresponding group without PEG modification.
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and reversible, but not mimicked by NAC. (A) LN-229 cells were incubated with MNPs (100 mg ml1; shaded area) with the magnet (Mag) underneath for 5 min or 2 h after addition of MNPs. Doubleheaded arrows indicate duration of EGCG treatment in groups (1)–(4). (B) LN-229 cells were incubated with MNPs (100 mg ml1) and NAC in the presence (+) or absence () of the magnet for 24 h. The antioxidant activity of EGCG and NAC was determined by the DPPH method (inset). Data are presented as mean SE (n ¼ 4–8). *: P < 0.05 compared with corresponding values without EGCG or NAC; †: P < 0.05 compared with corresponding values with Mag (5 min) or Mag (); §: P < 0.05 compared with the corresponding group with EGCG pre-treatment.
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2 to 6 h did not further enhance MNPcell. Similar effects were also observed for C6 cells (data not shown). NAC, with an antioxidant property similar to that of EGCG, did not exert an enhancement effect on MNP uptake (Fig. 4B), suggesting that the enhancement effect of EGCG may not be contributed to by its antioxidant property. To determine whether the enhancement effect of EGCG on MNP internalization may be predominantly mediated by endocytosis, experiments were conducted at 4 C. The application of magnetic force for 1 h signicantly enhanced MNPcell at 4 C, but not at 37 C. EGCG enhanced MNPcell by 1.6- and 3.6fold at 37 C in the presence of the magnet for 5 min and 1 h, respectively. At 4 C, the enhancement effect of EGCG was attenuated by 51% with the application of the magnet for 1 h, but not with the application of the magnet for 5 min (Fig. 5A). With confocal microscopy, internalization of MNPs was observed at 37 C, which was greatly enhanced by EGCG. Although MNPcell was not reduced by low temperature with 5 min of magnetic inuence (Fig. 5A), MNP internalization was not evident at 4 C (Fig. 5B). Quantitative results in response to EGCG treatment in Fig. 5A are modelled in Fig. 5C. In addition, EGCG may affect cell membrane uidity,35,36 which mediates the enhancement effects on MNP internalization. However, the concentration of EGCG used in the current study did not exert any effect on membrane uidity, as determined by membrane anisotropy (Fig. 5A, inset). Nevertheless, benzyl alcohol, an agent known to increase membrane uidity, decreased anisotropy but exerted no effect on cellular uptake of MNPs for all conditions studied (Fig. 5A), suggesting that the enhancement effect of EGCG may not be mediated by alteration of membrane uidity. To determine whether the enhancement effect of EGCG may be associated with modication of the surface characteristics of MNPs, effects of EGCG adsorption on MNPcell were examined. EGCG adsorption was performed by incubation of EGCG (1.1 mM) and MNP–PEG (5 mg ml1), followed by magnetic separation. Table 1 shows the size and zeta potential of EGCG-adsorbed vs. pristine MNPs. The adsorption of EGCG did not signicantly alter the size of the MNPs, however, EGCG adsorption altered the zeta potential of MNPs and MNP–PEG to approximately 30 mV. To determine the stability of MNP– EGCG composite, the antioxidant activity of EGCG associated with MNPs was determined. Aer magnetic separation, the antioxidant activity of EGCG was primarily associated with the composite aer storage at 4 C for 1 to 24 h; the antioxidant activity in the supernatant solution was only 2.4–5.5% of that associated with the particles aer magnetic separation. These results demonstrated that adsorbed EGCG remained stable with the MNPs during the 24 h period (Fig. 6A). In all groups studied with LN-229 cells, application of a magnetic eld for 24 h signicantly enhanced MNP uptake by 1.4- to 9.6-fold (P < 0.05; Fig. 6B). With the same amount of EGCG of 6.4 mM, MNP uptake enhancements induced by MNP-associated EGCG (bound) were 10.3 and 2.5 times that induced by free EGCG in the absence and presence of the magnet, respectively (P < 0.05). With EGCG concentration of 12.8 mM, bound EGCG-induced enhancing effect on MNP uptake was 8.1 and 1.1 times that induced by free
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Fig. 5 EGCG increased cell-associated MNPs at 4 C and 37 C without altering the fluidity of plasma membrane. C6 (A) or LN-229 (B) cells were incubated at 4 vs. 37 C for 20 min before administration of MNPs (A) or CMX–MNP (B) with EGCG (10 mM) or benzyl alcohol (15– 30 mM), followed by additional 1 h (A and B) or 15 min (A, inset) incubation. After the treatment, the cells were counterstained with rhodamine phalloidin (red) and DAPI (blue) prior to confocal microscopy (B). The schematic diagrams (C) illustrate the relative amount of cell-associated MNPs (MNPcell) measured in (A). Values are presented as mean SE (n ¼ 4–8). *: P < 0.05 compared with corresponding vehicle group; #: P < 0.05 compared with corresponding group at 4 C; §: P < 0.05 compared with the corresponding group with 5 min magnetic field.
EGCG in the absence and presence of the magnet, respectively (P < 0.05). Similar results were also observed in culture of HUVEC in response to EGCG (Fig. 6C). Adsorption of EGCG enhanced MNP uptake by 6.7- and 8.3-fold in the absence and presence of the magnet, respectively; adsorption of EGCG
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Table 1
Nanoscale Size and zeta potential of MNPsa
Particle
Size (nm)
Zeta potential (mV)
MNP MNP-PEG MNP-EGCG MNP-PEG-EGCG
263.7 5.5 299.8 3.6 270.3 6.3 296.3 4.0
40.1 11.3 25.5 31.9
a
0.3 0.4 0.1 0.5
Data presented as mean SE, n ¼ 3.
enhanced MNP–PEG uptake by 7- and 20-fold in the absence and presence of the magnet, respectively. These ndings suggest that EGCG adsorption may account for the enhanced uptake of MNPs. The results of the MTT assay demonstrated that there was no signicant cytotoxicity in the groups studied except for MNP-EGCG (Fig. 7), which was probably because of a higher concentration of EGCG in the cell surroundings with the MNP–EGCG group in the presence of the magnet.
Discussion Nanoparticles have been tested as drug carriers in therapeutics.1,14,38 Interaction of the carriers with a biological system may determine the efficacy of the pharmacological treatment. In this study, we have demonstrated a novel effect of food components: tea catechins, on cellular uptake of nanoparticles. The enhancement effects of EGCG on cellular uptake of MNPs appear to be cell-specic, concentration-dependent, and synergized with magnetic force application. Our study also reveals a potential food–drug interaction that may have an impact on pharmacokinetics and toxicity of nanocarriers. The results demonstrate that EGCG signicantly increased MNPs in cytoplasm, which were co-localized with lysosomes as observed using confocal microscopy, suggesting that MNPs were internalized into cytoplasm via endocytosis. The imaging results were consistent with those from ow cytometry and KSCN quantication of iron, and were in agreement with those reported in the literature7–9,39–42 demonstrating that the main route of nanoparticles entering cells was via endocytosis, both in glioma39,42 as well as in normal cells.40,41 Although the colorimetric quantication of iron may not allow differentiation of MNPs in the cytoplasm from those on the plasma membrane,21 the assay provides quantitative determination of cell-associated MNPs (MNPcell), which reveal the consequence of particle–plasma membrane interaction. Whether EGCG exerts any effect on particles escaping from lysosomes or exocytosis remains to be determined. Our results demonstrate that EGCG exerted a signicant enhancement effect on MNPcell even at 4 C (Fig. 5A) when energy-dependent endocytosis was blocked.43 Thus, the effect of EGCG may be mediated by, at least in part, a mechanism other than endocytosis. Similar effects were observed aer application of magnetic force at 4 C for one hour, which is consistent with a recent observation that magnetic eld may increase MNP attachment to the cells at 4 C.44 It is likely that EGCG may increase MNPcell by enhancing the adsorption of MNPs to the
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Adsorption of EGCG enhanced MNP uptake by glioma and endothelial cells. The MNP–PEG–EGCG complex was produced by incubation of EGCG and MNPs or MNP–PEG at 37 C for 2 h. The stability of the complex at 4 C was determined by measuring the antioxidant activity of EGCG associated with the particles (A). The effects of free (f) vs. bound (b) forms of EGCG (6.4 and 12.8 mM) with MNPs (100 mg ml1) in LN-229 cells (B) and the effects of EGCG (10 mM) adsorption on uptake of MNPs or MNP–PEG (140 mg ml1) by HUVEC (C) for 24 h were determined. Values are presented as mean SE. §: P < 0.05 compared with corresponding MNP–PEG–EGCG values; *: P < 0.05 compared with corresponding groups without magnetic field; †: P < 0.05 compared with the corresponding free form groups; #: P < 0.05 compared with the corresponding group with lower EGCG concentration or without EGCG. Fig. 6
plasma membrane of glioma cells. In addition, magnetic force may further increase MNP attachment to the plasma membrane in the presence of EGCG, as demonstrated by a synergistic effect of magnetic force and EGCG (Fig. 5C). However, endocytosis may play a pivotal role in the synergistic effects of EGCG and magnetic force, because the value of MNPcell doubled at 37 C vs. 4 C in the presence of both EGCG
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Fig. 7 Cytotoxicity of MNPs/MNP–PEG with free or bound EGCG under the influence of magnetic field for 5 min vs. 2 h. LN229 cells were incubated with MNPs or MNP–PEG (100 mg ml1) in the presence of free (f) or bound (b) form of EGCG (9.5 mM) for 2 h. Values are presented as mean SE (n ¼ 6). *, P < 0.05 compared with the control group without nanoparticles or EGCG; †, P < 0.05 compared with the corresponding groups with 5 min magnetic field.
and the magnet. Thus, the enhancement effect of EGCG at 37 C may be mediated by increasing both membrane-bound and internalized MNPs (Fig. 5C). The interaction of MNPs and plasma membrane appears to be strong enough to be retained aer the PBS wash, if not internalized. Previous studies have demonstrated that nanoparticles may induce membrane perturbation.45,46 Such membrane ruffling may provide a physical barrier during the wash procedure and be responsible, at least in part, for an increased MNPcell in response to EGCG or magnetic force. When endocytosis was blocked by low temperature, the enhancement effect of EGCG may be because of chemical interaction of MNPs and plasma membrane, suggesting that EGCG may modify the surface characteristics of MNPs. In addition, the enhancing effect of EGCG was not observed when EGCG was removed during administration of MNPs to the culture (Fig. 4A), suggesting that the co-existence of EGCG and MNPs appears to be required for EGCG to have its enhancing effects. Although EGCG may bind to proteins on the plasma membrane and activate certain signaling pathways,24,26 the enhancing effects on MNP uptake appear to be acute and reversible, which are independent of signaling pathways that require protein phosphorylation or gene expression. It is likely that the enhancing effect of EGCG might be a result of a direct interaction of EGCG with plasma membrane of tumor cells. It has been reported that nanoparticles with a hydrophobic surface may interact more readily with the lipid bilayer of the plasma membrane.12,13,47 EGCG exerts a calculated octanol–water partition coefficient (log P) of 2.29, suggesting that it has hydrophobic characteristics. Previous studies have also reported that EGCG and other catechins may form hydrogen bonds with lipid bilayers.48,49 Alternatively, EGCG may bind to membrane receptors, including a 67 kDa laminin receptor,24,31,32 or specic membrane regions such as lipid ras.24,50 All the above mentioned mechanisms of EGCG–membrane interaction may contribute to the enhancing effects of EGCG on MNP uptake. In addition, the zeta potential of MNPs is one of many factors that may affect how MNPs interact with cells. The zeta potential
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changes to the range of 25 to 32 mV aer EGCG adsorption in distilled water, demonstrating the effect of EGCG on the surface characteristics of the MNPs. Nevertheless, previous studies have shown that the zeta potential of nanoparticles was between 9 and 19 mV aer incubation in serum-supplemented culture medium despite the original zeta potential,51,52 which is probably due to the masking effect of serum proteins in the medium. Therefore, the enhancing effect of EGCG on MNP uptake is unlikely to be the result of the contribution of changes of zeta potential in the culture medium. The enhancing effect on MNP cellular uptake was also observed aer EGCG modication of MNPs via adsorption and magnetic separation. With the same concentration of EGCG in culture, greater MNPcell was observed with the bound vs. free form of EGCG (Fig. 6B), which was probably because of more EGCG coming into contact with the plasma membrane of the cell following MNP sedimentation. In spite of poor bioavailability of tea catechins,26,53 oral consumption of tea preparations may achieve a peak plasma concentration of 5–7 mM,26 which is within the range of concentrations for EGCG that exerted an enhancing effect in the current study. As shown in Fig. 6B, EGCG-bound MNPs had a signicantly higher MNPcell. It is thus anticipated that MNP–catechin interactions may allow MNP retention at cell surroundings with local concentration of catechins much higher than that in plasma. However, whether the enhanced MNP–plasma membrane interaction may occur in tumors remains to be determined. The effect of EGCG was cell-specic, because no effect was observed on MNP uptake by HUVEC without a magnetic eld. Nevertheless, the magnetic force signicantly enhanced MNP uptake by HUVEC, especially for MNPs not modied by PEG. The underlying mechanism that causes cell specicity is uncertain. Whether the difference may be because of characteristics of the plasma membrane of normal vs. tumor cells remains to be determined. Nevertheless, PEG modication may jeopardize particle internalization by the target cells, but the uptake of PEG-modied MNPs may be restored by co-administration of EGCG and a magnetic eld. The effects of PEG on tumor cell uptake of nanoparticles are consistent with the previous ndings that lower transfection efficiency was associated with carriers modied by PEG.54 It has also been demonstrated that EGCG may be incorporated into a nanocarrier composite for treatment of prostate cancer in vitro55 and in animal models.31 Although EGCGinduced growth inhibition may play a role in the previously mentioned studies, it may be that EGCG can augment the therapeutic effects by enhancing nanoparticle uptake in these systems. The current studies may provide a novel interpretation of the effect of EGCG in the nanocarrier systems for treatment of cancer.
Conclusions We demonstrated a feasible application of a food component in nanocarrier systems for targeted delivery of drugs or biological molecules by MNPs. Combined application of EGCG and a magnetic eld may markedly increase MNP uptake, which may
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lead to better therapeutic or transfection efficacy. In addition, MNP–EGCG composites may be produced by physical adsorption, which is a simple approach to facilitate tumor uptake of MNPs while sustaining the bioactivity in circulation. With cell specicity, the targeting effects of the system may also be augmented, which allows extensive tumor uptake but spares the non-targets, such as vascular endothelial cells. Combined application of magnetic force and EGCG may facilitate particle internalization in targeted therapeutics or magnetofection.
Acknowledgements We thank the Ministry of Science and Technology of the Republic of China (NSC 100-2120-M-182-001- and NSC 1012320-B-182-038-), Healthy Aging Research Program at Chang Gung University (EMRPD1A0841), and Chang-Gung Memorial Hospital (CMRPD1D0231) for funding support. The authors thank Dr Ying-Tung Lau at Chang Gung University of Science and Technology for assistance in HUVEC culture, and Dr HawMing Huang at Taipei Medical University for assistance in uorescence anisotropy measurement.
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