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Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells
Acta Biomaterialia xxx (2013) xxx–xxx
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Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells Viktoriya Sokolova a, Diana Kozlova a, Torben Knuschke b, Jan Buer b, Astrid M. Westendorf b, Matthias Epple a,⇑ a b
Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaets str. 5-7, D-45117 Essen, Germany Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Hufeland str. 55, 45147 Essen, Germany
a r t i c l e
i n f o
Article history: Received 21 December 2012 Received in revised form 15 February 2013 Accepted 19 February 2013 Available online xxxx Keywords: Calcium phosphate Nanoparticles Endocytosis Inhibitors
a b s t r a c t The uptake of calcium phosphate nanoparticles (diameter 120 nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3 h of incubation. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles are widely used in cell biology and medicine for transfection, gene silencing, photodynamic therapy and drug delivery [1–8]. Large molecules alone often cannot penetrate the cell membrane; therefore, an efficient carrier is needed [9,10]. It was shown that nanoparticles with a size up to several hundred nanometers can enter the cells in membrane-bound vesicles by endocytosis [11]. Endocytosis consists of three major steps: formation of membrane vesicles with the cargo, endosomal delivery of the cargo inside the cell and the distribution to various organelles inside the cell [9,11–14]. It is generally distinguished between clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin- or caveolin-independent endocytosis and macropinocytosis (Fig. 1) [12,15]. Calcium phosphate nanoparticles are well known as nanoscopic mineral of bone [16–18], and are also used as carriers for the transport of genes (DNA; siRNA) [8,19,20] and drugs [21,22] into cells. Calcium phosphate is also used as bone substitution material as a paste in the form of water-dispersed nanoparticles [23,24]. Concerns have been raised about the potential problems of an increased intracellular calcium level after cellular uptake [25,26]. A cytotoxic increase of the intracellular calcium level was observed for DNA/calcium phosphate precipitates [27] which are formed with the classical calcium phosphate transfection method [28]. ⇑ Corresponding author. Tel.: +49 201 2402; fax: +49 201 1832621. E-mail address: [email protected] (M. Epple).
However, in the case of functionalized calcium phosphate nanoparticles in the colloidal state, no adverse effects were found [27], probably due to the much lower dose of calcium in this case. If calcium phosphate nanoparticles are used to deliver biomolecules into a cell, a cellular uptake is necessary. Thus, it is important to address the question of the uptake mechanism and the final fate of the nanoparticles within a cell. Here we studied the uptake of fluorescent calcium phosphate nanoparticles with a diameter of 120 nm, both cationic and anionic, to provide a better understanding of the mechanisms and in turn to optimize their application as gene and drug delivery vehicles. Their intracellular pathway was also followed by covalent functionalization with the pHsensitive dye SNARF-1 (carboxyseminaphthorhodafluor-1).
2. Materials and methods 2.1. Preparation of anionic triple-shell CaP/CpG/CaP/CpG nanoparticles The synthesis was carried out as described previously [29]. Aqueous solutions of calcium nitrate (6.25 mM; Merck p.a.) and diammonium hydrogen phosphate (3.74 mM; Merck p.a.) were rapidly mixed by pumping them into a glass vessel. The pH of both solutions was adjusted beforehand to 9 with NaOH (0.1 M; Merck, p.a.). A few seconds after mixing, 1 ml of the calcium phosphate nanoparticle dispersion was taken with a syringe and mixed with 0.2 ml of a 1:3 mixture of fluorescent CpG (labelled either with Alexa 488 or with Alexa 555) and non-fluorescent CpG (total
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.02.034
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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Fig. 1. Schematic representation of the different internalization mechanisms for nanoparticles, including clathrin-mediated endocytosis, caveolin-mediated endocytosis and macropinocytosis.
concentration of CpG 0.4 mg ml1). The oligonucleotides CpG, CpGAlexa 488 and CpG-Alexa 555 were obtained from Life Technologies (Germany) and had the sequence 50 -TCCATGACGTTCCTGAC GTT-30 . To add the second layer (calcium phosphate), 0.5 ml of an aqueous solution of Ca(NO3)2 (6.25 mM) and 0.5 ml of an aqueous solution of (NH4)2HPO4 (3.74 mM) were consecutively added. For the third layer (CpG), 0.2 ml of a 1:3 mixture of fluorescent CpG and non-fluorescent CpG was added. The functionalized triple-shell CaP/CpG/CaP/CpG nanoparticles were collected by ultracentrifugation for 30 min at 66,000g. The supernatant was taken with a syringe to quantify the amount of Ca2+ by atomic absorption spectroscopy (AAS). The concentration of Ca2+ in the supernatant was 50.4 lg ml1. Because the theoretical concentration of Ca2+ in the triple-shell nanoparticles was 104 lg ml1, 53.6 lg ml1 of Ca2+ were still present in the particles and 2.4 ml 53.6 lg ml1 = 128.6 lg Ca, corresponding to 323 lg calcium phosphate (calculated as hydroxyapatite; Ca5(PO4)3OH) were incorporated in the nanoparticles. The particles were dispersed in the original volume of water (2.4 ml) by ultrasonication (UP50H, Hielscher, Ultrasound Technology; sonotrode 3, cycle 0.8, amplitude 60%, 10 s). The final concentration of calcium phosphate was therefore 323 lg/2.4 ml = 135 lg ml1. 2.2. Preparation of cationic CaP/PEI/SiO2/NH-FITC and CaP/PEI/SiO2/ NH-SNARF nanoparticles
amplitude 70%, 15 s). The covalent functionalization of CaP/PEI/ SiO2 nanoparticles with amino groups was carried out as follows: 50 ll (3-aminopropyl)triethoxysilane (APTES; Sigma–Aldrich) were dissolved in 40 ml ethanol. 10 ml of the CaP/PEI/SiO2 nanoparticle dispersion was added, and the mixture was stirred for 8–10 h at room temperature. Then the particles were dispersed in 10 ml ethanol under ultrasonication as described above. 100 ll of fluorescein isothiocyanate (FITC; Merck), dissolved in ethanol (1 mg ml1), were added to the amino-functionalized nanoparticles for the covalent attachment of FITC. After stirring at room temperature for 18–20 h, the particles were purified several times by consecutive ultracentrifugation and redispersion in pure water by ultrasonication as described above to remove unreacted reagents. The final volume after redispersion was 7 ml of water. The concentration of calcium in the nanoparticle dispersion was determined to 82.2 lg ml1 by AAS, corresponding to 206 lg ml1 calcium phosphate (as hydroxyapatite). For covalent attachment of SNARF-1 (carboxylic acid, acetate, succinimidyl ester; Life Technologies), we added 100 ll of SNARF1 DMSO solution (0.5 mg ml1) to 10 ml of the dispersion of CaP/PEI/SiO2/NH2 nanoparticles. Then the mixture was stirred for 18–20 h at room temperature and purified by ultrasonication/redispersion as described above for FITC. We assumed the same nanoparticle concentration as with FITC-conjugated nanoparticles because of the identical synthetic route. 2.3. Characterization
Polyethylenimine-stabilized calcium phosphate nanoparticles (CaP/PEI) were prepared as previously described [30]. Aqueous solutions of calcium lactate (18 mM, pH 10), (NH4)2HPO4 (10.8 mM, pH 10) and PEI (2 g l1; Sigma–Aldrich; branched; 25 kDa) were simultaneously pumped in a volume ratio of 5 ml: 5 ml: 7 ml into a stirred glass vessel containing 20 ml of ultrapure water during 1 min at room temperature. After 20 min stirring, 10 ml of the CaP/PEI-nanoparticle dispersion was added to a mixture of 40 ml ethanol, 50 ll tetraethylorthosilicate (TEOS; Sigma– Aldrich) and 26 ll aqueous ammonia solution (30–33%) for coating with a thin layer of silica. This reaction mixture was stirred for 16 h at room temperature. Then the particles were isolated by ultracentrifugation and redispersed in the original volume of water (10 ml) (UP50H, Hielscher, Ultrasound Technology; sonotrode 7, cycle 0.8,
Scanning electron microscopy was performed with an ESEM Quanta 400 instrument with gold/palladium-sputtered samples. Dynamic light scattering and zeta potential determinations were performed with a Zetasizer nanoseries instrument (Malvern Nano-ZS, laser: k = 532 nm) using the Smoluchowski approximation and taking the data from the Malvern software without further correction. The particle size data refer to scattering intensity distributions (z-average). Ultracentrifugation was performed at 25 °C with an Optima XL-I instrument (Beckman–Coulter). The cells were analysed by flow cytometry with a LSR II instrument using the DIVA software (BD Biosciences). Confocal laser scanning microscopy was performed with a Zeiss LSM 510 Axiovert 200 instrument.
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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V. Sokolova et al. / Acta Biomaterialia xxx (2013) xxx–xxx Table 1 Calculated number of dye molecules (CpG and FITC, respectively) per calcium phosphate nanoparticle. Sample
Number of nanoparticles per m3 colloidal dispersion
Number of dye molecules per m3 colloidal dispersion
Number of dye molecules per nanoparticle
CaP/CpG/CaP/CpG CaP/PEI/SiO2/NHFITC
4.75 1016 7.25 1016
6.62 1020 9.76 1021
13,900 135,000
Fig. 2. Schematic representation of the conjugation of SNARF-1 molecules to amino groups on the nanoparticle surface.
2.4. Calculation of the amount of dye per calcium phosphate nanoparticle The nanoparticle concentration was calculated by taking the concentration of calcium phosphate in the dispersion and assuming spherical nanoparticles with a diameter of 120 nm. The number of dye molecules was determined by quantitative UV spectroscopy after previous calibration for CpG-Alexa 555 and FITC, respectively (see Eq. (1) and Table 1):
NðNPÞ ¼
mðCapÞ 3 mðCaPÞ ¼ mðNPÞ 4 p rðNPÞ3 qðCaPÞ
Table 2 Dynamic light scattering (DLS) data of water-dispersed calcium phosphate nanoparticles (PDI: polydispersity index). Sample
Hydrodynamic diameter (nm)
PDI
Zeta potential (mV)
CaP/CpG/CaP/CpG CaP/PEI/SiO2/NHFITC CaP/PEI/SiO2/NHSNARF
322 183
0.348 0.242
25 ± 4 +21 ± 4
195
0.356
+22 ± 5
ð1Þ
NðdyeÞ ¼ cðdyeÞ NA 3
where N(NP) is the number of nanoparticles per m ; m(CaP) is the mass of all synthesized calcium phosphate nanoparticles per m3 with m(CaP/CpG/CaP/CpG) = 0.135 kg m3 and m(CaP/PEI/SiO2/NH-FITC) = 0.206 kg m3, respectively; m(NP) is the mass of one calcium phosphate nanoparticle: m(NP) = 2.84 1018 kg; r(NP) is the average radius of one nanoparticle: r(NP) = 60 109 m; q(CaP) is the density of calcium phosphate: q(CaP as hydroxyapatite) = 3140 kg m3; N(dye) is the number of dye molecules per m3; c(dye) is the concentration of the dye in the nanoparticle dispersion: c(CpG-Alexa 555) = 0.00780 kg m3/(7.1176 kg mol1) = 0.00110 mol m3 and c(FITC) = 0.00631 kg m3/(0.3894 kg mol1) = 0.0162 mol m3, respectively; and NA is the Avogadro constant: NA = 6.022 1023 mol1. 2.5. Nanoparticle uptake HeLa cells (human epithelial cervical cancer cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS) at 37 °C and 10% CO2 and subcultivated according to standard cell culture protocols. 12 h prior to uptake experiments, the cells were trypsinized and seeded in six-well plates with 1 105 cells per well in 2 ml DMEM with FCS. The following final concentrations of calcium phosphate nanoparticles per well
were tested: 2.30 109 ml1, 3.57 109 ml1, 5.17 109 ml1, 6.65 109 ml1 for CaP/CpG/CaP/CpG and 0.072 109 ml1, 0.18 109 ml1, 0.72 109 ml1, 1.77 109 ml1 for CaP/PEI/ SiO2/NH-FITC nanoparticles. The cells were incubated for 3 h, then the whole cell culture medium was removed. The cells were washed three times with phosphate-buffered saline (PBS). Afterwards, the cells were trypsinized, centrifuged and resuspended in 200 ll of FACS solution. The cells were then analysed by flow cytometry. 2.6. Immunofluorescence and localization of nanoparticles in the cell HeLa cells were cultured in DMEM, supplemented with 10% FCS at 37 °C and 10% CO2 and subcultivated according to standard cell culture protocols. 12 h prior to the uptake experiments, the cells were trypsinized and seeded in cell culture dishes with 5 104 cells per well in 0.5 ml DMEM with FCS. The incubation with either cationic or anionic nanoparticles was carried out as follows. Either 40 ll (corresponding to 3.57 109 nanoparticles ml1 after dilution) of CaP/CpG/CaP/CpG nanoparticles or 5 ll (corresponding to 0.72 109 nanoparticles ml1 after dilution) of CaP/PEI/SiO2/NH-SNARF nanoparticles were added, the cells were incubated for 3 h and then the whole cell culture medium was removed. The cells were washed three
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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Fig. 3. Scanning electron micrographs of negatively charged CaP/CpG/CaP/CpG nanoparticles and of positively charged CaP/PEI/SiO2/NH-dye nanoparticles (dye = FITC or SNARF), together with a schematic representation of their multi-shell structure.
Fig. 4. Functionalized calcium phosphate nanoparticles are increasingly taken up by cells when their concentration increases. The cells were incubated with different amounts of anionic CaP/CpG/CaP/CpG nanoparticles (labelled with Alexa488; (A)) and cationic CaP/PEI/SiO2/NH-FITC nanoparticles (B) for 3 h at 37 °C. Mock: cells without nanoparticles.
times with PBS to remove adhering nanoparticles. Therefore, only nanoparticles which were either taken up by the cells or strongly adsorbed onto the cellular surface remained. Afterwards, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then washed three times with PBS. Then the cells were stained for 15 min with 1 ll of anti-LAMP-1 staining reagent (mouse anti-human CD107A/LAMP-1 FITC conjugate; Life Technologies) for lysosomes (0.5 lg ml1) and washed three times with PBS. The cells were then stained with DAPI for the cell nucleus
and washed three times with PBS. Finally, the cells were studied by confocal laser scanning microscopy. The cellular uptake kinetics of CaP/PEI/SiO2/NH-SNARF nanoparticles by HeLa cells was studied as follows. The incubation was performed for 3, 5, 13 and 25 h with 5 ll (0.72 109 nanoparticles ml1 after dilution) of CaP/PEI/SiO2/NH-SNARF nanoparticles in cell culture dishes with 5 104 cells per well in 0.5 ml DMEM with FCS. After these times, the whole cell culture medium was removed, and the cells were washed three times with PBS. Then the
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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Fig. 5. MTT test of HeLa cells treated with different amounts of anionic CaP/CpG/CaP/CpG nanoparticles (labelled with Alexa488; (A)) and cationic CaP/PEI/SiO2/NH-FITC nanoparticles (B). Mock: cells without nanoparticles.
Fig. 6. FACS data of the cellular uptake of negatively charged calcium phosphate nanoparticles (CaP/CpG/CaP/CpG; labelled with Alexa 488) at 4 °C and in the presence of various inhibitors of endocytosis. Wortmannin and LY294002 efficiently blocked the nanoparticle uptake, indicating macropinocytosis as uptake mechanism.
cells were stained with DAPI (40 ,6-diamidino-2-phenylindole dilactate), washed three times with PBS and studied by confocal laser scanning microscopy.
solution and analysed by flow cytometry. Statistical analysis of the mean fluorescence for each treatment compared to the untreated cells (mock) was done using ANOVA in Sigmaplot.
2.7. Blocking nanoparticle uptake using inhibitors of endocytosis
2.8. Cytotoxicity of nanoparticles
Cells were plated in six-well plates with 105 cells per well in 2 ml DMEM with FCS 1 day before the uptake experiments. The cells were pre-incubated with various inhibitors: wortmannin (100 ng ml1), LY294002 (20 lg ml1), nocodazole (10 lg ml1), chlorpromazine (1 lg ml1) and nystatin (10 lg ml1) for 30 min, followed by the addition of CaP/CpG/CaP/CpG (5.17 109 nanoparticles ml1 after dilution) or CaP/PEI/SiO2/NH-FITC (0.72 109 nanoparticles ml1 after dilution) and incubation for 3 h. The cells were then washed three times with PBS (4 °C). Afterwards, the cells were trypsinized, centrifuged, resuspended in 200 ll of FACS
The cell viability was analysed by an MTT assay 3 h after the incubation with the following concentrations of calcium phosphate nanoparticles per well after dilution: 2.30 109 ml1, 3.57 109 ml1, 5.17 109 ml1, 6.65 109 ml1 for CaP/CpG/CaP/CpG nanoparticles and 0.072 109 ml1, 0.18 109 ml1, 0.72 109 ml1, 1.77 109 ml1 for CaP/PEI/SiO2/NH-FITC nanoparticles under the same conditions as in the uptake studies described above. MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Sigma, Taufkirchen, Germany) was dissolved in PBS (5 mg ml1) and then diluted to 1 mg ml1 in the cell culture medium. The cell culture
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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Fig. 7. The uptake of calcium phosphate nanoparticles in the presence of endocytosis inhibitors, normalized to the uptake in the absence of inhibitors (100%). (A) CaP/CpG/ CaP/CpG (anionic; labelled with Alexa488); (B) CaP/PEI/SiO2/NH-FITC (cationic). ( means a significance of P < 0.05).
Fig. 8. Confocal laser scanning microscopy of HeLa cells after 3 h of incubation with negatively charged CaP/CpG/CaP/CpG nanoparticles ((labelled with Alexa555; (I)) and positively charged CaP/PEI/SiO2/NH-SNARF nanoparticles (II). (A) DAPI staining; (B) anti-LAMP-1 staining for lysosomes; (C) red fluorescent nanoparticles; (D) merged image; the yellow colour indicates a colocalization of green anti-LAMP-1 and red fluorescing nanoparticles. Scale bar = 5 lm.
medium of the incubated cells was replaced by 300 ll of the MTT solution and incubated for 1 h at 37 °C under 5% CO2 in humidified atmosphere. 300 ll DMSO were added to the cells. After 30 min, a 100 ll aliquot was taken for spectrophotometric analysis with a Multiscan FC instrument (Thermo Fisher scientific, Vantaa, Finland) at k = 570 nm. The absorption of incubated cells was normalized to that of control (untreated) cells, thereby indicating the relative level of cell viability.
3. Results and discussion Positively charged calcium phosphate nanoparticles were prepared by covalent functionalization as described earlier [30]. FITC was attached by reaction of the amino groups on the particle surface with the isothiocyanate group of FITC [30], giving CaP/PEI/ SiO2/NH-FITC nanoparticles. SNARF-1 was conjugated by a reaction between the amino groups on the particle surface with the succinimidyl-groups of functionalized SNARF-1 molecules (Fig. 2), giving CaP/PEI/SiO2/NH-SNARF nanoparticles. Negatively charged triple-
shell nanoparticles were prepared by multi-step precipitation, giving CaP/CpG/CaP/CpG nanoparticles. All functionalized calcium phosphate nanoparticles were characterized by dynamic light scattering and scanning electron microscopy (Table 2). A spherical morphology of the nanoparticles with a diameter of 120 nm was shown by scanning electron microscopy (SEM; Fig. 3). To follow the uptake of calcium phosphate nanoparticles into cells, we incubated HeLa cells with different concentrations of nanoparticles (2.30–6.65 109 nanoparticles ml1 of CaP/CpG/ CaP/CpG (labelled with Alexa488) and 0.072–1.77 109 nanoparticles ml1 of CaP/PEI/SiO2/NH-FITC). The resulting fluorescence per cell, which is a measure of the amount of uptaken nanoparticles, was determined by flow cytometry. As shown in Fig. 4, a higher concentration of fluorescent calcium phosphate nanoparticles led to an increased uptake and in turn to a larger average fluorescence per cell. The viability of cells treated with increasing amounts of calcium phosphate nanoparticles was assessed by the MTT test. The viability of HeLa cells after incubation with negatively or positively charged nanoparticles for 3 h was 90–100% and did not depend
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Fig. 9. Confocal laser scanning microscopy (CLSM) of HeLa cells after incubation with positively charged CaP/PEI/SiO2/NH-SNARF nanoparticles for 3 h, 5 h, 13 h and 25 h, respectively. (A) DAPI-channel (cell nucleus), (B) FITC-channel (nanoparticles at low pH in lysosomes), (C) TRITC-channel (nanoparticles at neutral pH in endosomes) and (D) merged image. Mock: cells without nanoparticles.
on the concentration (Fig. 5). We conclude that neither kind of nanoparticles was cytotoxic. The route of entry of nanoparticles into the cell and their final intracellular localization is decisive for a potential application as gene or drug delivery agent. We have used several inhibitors of endocytosis to elucidate the mechanism of the cellular uptake of negatively and positively charged calcium phosphate nanoparticles. HeLa cells were incubated for 30 min with the inhibitors and then treated either with CaP/CpG/CaP/CpG (anionic) or with CaP/PEI/SiO2/NH-FITC (cationic) nanoparticles. As a control, we used cells that had been incubated with nanoparticles in the absence of inhibitors. As negative control, the cells were incubated with nanoparticles at 4 °C for 3 h. As endocytosis is an energyconsuming process for the cell, it is generally inhibited at low temperature [12]. A decrease in the measured nanoparticle fluorescence, compared to the control, indicates the involvement of the specific endocytotic mechanism targeted by that inhibitor (Fig. 6). A statistically significant inhibition of the uptake of negatively charged nanoparticles was observed for wortmannin and
LY294002 (Fig. 7A). Therefore, the inhibition of uptake by wortmannin and LY294002 suggests that nanoparticles enter the cell by macropinocytosis [31,32]. A slight decrease in the uptake was induced by nocodazole for CaP/CpG/CaP/CpG nanoparticles. For positively charged calcium phosphate nanoparticles, a significant inhibition of the uptake was found in the case of wortmannin (Fig. 7B). No reduction in uptake with both types of nanoparticles was observed for either chlorpromazine or nystatin, which inhibit clathrin- or caveolin-mediated endocytosis, respectively [33,34]. Together these data show that the uptake of calcium phosphate nanoparticles into HeLa cells occurs by macropinocytosis rather than by clathrin- or caveolin-mediated endocytosis. The localization of nanoparticles was studied by confocal laser scanning microscopy. Endocytosis begins with the formation of membrane vesicles, continues with the transformation into early endosomes and their further maturation into late endosomes before fusion with lysosomes [35]. Lysosomal-associated membrane protein-1 (LAMP-1) was stained to visualize the lysosomes in the
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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cell. In Fig. 8 it is clearly shown that red-fluorescing nanoparticles are localized in lysosomes after 3 h of incubation. The intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport and endocytosis. pH-sensitive fluorophores have therefore been widely applied to monitor changes in the intracellular pH [36]. Among these pH-sensitive dyes, SNARF-1 gives a significant fluorescence emission shift from red to green when the pH is changed from neutral to acidic [37]. The local measurement of the pH after particle uptake sheds light on the localization inside the cell, e.g. within a lysosome with low pH [38–40]. SNARF-1-conjugated calcium phosphate nanoparticles were used for monitoring of the local pH around nanoparticles during their uptake [12]. CaP/PEI/SiO2/NH-SNARF nanoparticles emit red fluorescence at neutral pH whereas nanoparticles internalized by cells emit green fluorescence when they experience the acidic pH within an endosomal/lysosomal compartment [40]. Fig. 9 shows the fluorescence micrographs of HeLa cells after different incubation times with SNARF-1 pH-sensitive conjugated calcium phosphate nanoparticles. After 3 h of incubation, the nanoparticles start to enter lysosomes, as indicated by the color change of the nanoparticles from red to green. This process continues after 5 h and more. Note that in the endosomes/lysosomes, typically both colors (red and green) are located due to the presence of both forms of SNARF-1 at this moderately low pH (4 to 5). Under these conditions, calcium phosphate nanoparticles will be dissolved. This supports earlier reports that excessive concentrations of calcium phosphate (nano-)particles can lead to cell apoptosis due to an increased intracellular calcium level [25–27,41]. However, we have shown earlier that the released calcium is not harmful for the cells if moderate concentrations of calcium phosphate nanoparticles are used [27]. 4. Conclusions Fluorescent cationic and anionic calcium phosphate nanoparticles are increasingly taken up by cells when their concentration increases. The uptake pathway into HeLa cells is mainly macropinocytosis, leading to their localization first in endosomes and then in lysosomes. After 3 h, the nanoparticles have been taken up by cells and will start to dissolve under the acidic conditions in the lysosome. Thus, they are well suited as non-toxic carriers of all kinds of molecules across the cell membrane into the cytoplasm. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Transregio 60) to A.M.W., J.B. and M.E. We thank Prof. Dr. Eric Metzen and Melanie Baumann for help with confocal laser scanning microscopy. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 6, 8 and 9, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2013.02.034. References [1] Bausinger R, von Gersdorff K, Braeckmans K, Ogris M, Wagner E, Bräuchle C, et al. The transport of nanosized gene carriers unraveled by live-cell imaging. Angew Chem Int Ed 2006;45:1568–72.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells Viktoriya Sokolova a, Diana Kozlova a, Torben Knuschke b, Jan Buer b, Astrid M. Westendorf b, Matthias Epple a,⇑ a b
Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaets str. 5-7, D-45117 Essen, Germany Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Hufeland str. 55, 45147 Essen, Germany
a r t i c l e
i n f o
Article history: Received 21 December 2012 Received in revised form 15 February 2013 Accepted 19 February 2013 Available online xxxx Keywords: Calcium phosphate Nanoparticles Endocytosis Inhibitors
a b s t r a c t The uptake of calcium phosphate nanoparticles (diameter 120 nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3 h of incubation. Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles are widely used in cell biology and medicine for transfection, gene silencing, photodynamic therapy and drug delivery [1–8]. Large molecules alone often cannot penetrate the cell membrane; therefore, an efficient carrier is needed [9,10]. It was shown that nanoparticles with a size up to several hundred nanometers can enter the cells in membrane-bound vesicles by endocytosis [11]. Endocytosis consists of three major steps: formation of membrane vesicles with the cargo, endosomal delivery of the cargo inside the cell and the distribution to various organelles inside the cell [9,11–14]. It is generally distinguished between clathrin-mediated endocytosis, caveolin-mediated endocytosis, clathrin- or caveolin-independent endocytosis and macropinocytosis (Fig. 1) [12,15]. Calcium phosphate nanoparticles are well known as nanoscopic mineral of bone [16–18], and are also used as carriers for the transport of genes (DNA; siRNA) [8,19,20] and drugs [21,22] into cells. Calcium phosphate is also used as bone substitution material as a paste in the form of water-dispersed nanoparticles [23,24]. Concerns have been raised about the potential problems of an increased intracellular calcium level after cellular uptake [25,26]. A cytotoxic increase of the intracellular calcium level was observed for DNA/calcium phosphate precipitates [27] which are formed with the classical calcium phosphate transfection method [28]. ⇑ Corresponding author. Tel.: +49 201 2402; fax: +49 201 1832621. E-mail address: [email protected] (M. Epple).
However, in the case of functionalized calcium phosphate nanoparticles in the colloidal state, no adverse effects were found [27], probably due to the much lower dose of calcium in this case. If calcium phosphate nanoparticles are used to deliver biomolecules into a cell, a cellular uptake is necessary. Thus, it is important to address the question of the uptake mechanism and the final fate of the nanoparticles within a cell. Here we studied the uptake of fluorescent calcium phosphate nanoparticles with a diameter of 120 nm, both cationic and anionic, to provide a better understanding of the mechanisms and in turn to optimize their application as gene and drug delivery vehicles. Their intracellular pathway was also followed by covalent functionalization with the pHsensitive dye SNARF-1 (carboxyseminaphthorhodafluor-1).
2. Materials and methods 2.1. Preparation of anionic triple-shell CaP/CpG/CaP/CpG nanoparticles The synthesis was carried out as described previously [29]. Aqueous solutions of calcium nitrate (6.25 mM; Merck p.a.) and diammonium hydrogen phosphate (3.74 mM; Merck p.a.) were rapidly mixed by pumping them into a glass vessel. The pH of both solutions was adjusted beforehand to 9 with NaOH (0.1 M; Merck, p.a.). A few seconds after mixing, 1 ml of the calcium phosphate nanoparticle dispersion was taken with a syringe and mixed with 0.2 ml of a 1:3 mixture of fluorescent CpG (labelled either with Alexa 488 or with Alexa 555) and non-fluorescent CpG (total
1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.actbio.2013.02.034
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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V. Sokolova et al. / Acta Biomaterialia xxx (2013) xxx–xxx
Fig. 1. Schematic representation of the different internalization mechanisms for nanoparticles, including clathrin-mediated endocytosis, caveolin-mediated endocytosis and macropinocytosis.
concentration of CpG 0.4 mg ml1). The oligonucleotides CpG, CpGAlexa 488 and CpG-Alexa 555 were obtained from Life Technologies (Germany) and had the sequence 50 -TCCATGACGTTCCTGAC GTT-30 . To add the second layer (calcium phosphate), 0.5 ml of an aqueous solution of Ca(NO3)2 (6.25 mM) and 0.5 ml of an aqueous solution of (NH4)2HPO4 (3.74 mM) were consecutively added. For the third layer (CpG), 0.2 ml of a 1:3 mixture of fluorescent CpG and non-fluorescent CpG was added. The functionalized triple-shell CaP/CpG/CaP/CpG nanoparticles were collected by ultracentrifugation for 30 min at 66,000g. The supernatant was taken with a syringe to quantify the amount of Ca2+ by atomic absorption spectroscopy (AAS). The concentration of Ca2+ in the supernatant was 50.4 lg ml1. Because the theoretical concentration of Ca2+ in the triple-shell nanoparticles was 104 lg ml1, 53.6 lg ml1 of Ca2+ were still present in the particles and 2.4 ml 53.6 lg ml1 = 128.6 lg Ca, corresponding to 323 lg calcium phosphate (calculated as hydroxyapatite; Ca5(PO4)3OH) were incorporated in the nanoparticles. The particles were dispersed in the original volume of water (2.4 ml) by ultrasonication (UP50H, Hielscher, Ultrasound Technology; sonotrode 3, cycle 0.8, amplitude 60%, 10 s). The final concentration of calcium phosphate was therefore 323 lg/2.4 ml = 135 lg ml1. 2.2. Preparation of cationic CaP/PEI/SiO2/NH-FITC and CaP/PEI/SiO2/ NH-SNARF nanoparticles
amplitude 70%, 15 s). The covalent functionalization of CaP/PEI/ SiO2 nanoparticles with amino groups was carried out as follows: 50 ll (3-aminopropyl)triethoxysilane (APTES; Sigma–Aldrich) were dissolved in 40 ml ethanol. 10 ml of the CaP/PEI/SiO2 nanoparticle dispersion was added, and the mixture was stirred for 8–10 h at room temperature. Then the particles were dispersed in 10 ml ethanol under ultrasonication as described above. 100 ll of fluorescein isothiocyanate (FITC; Merck), dissolved in ethanol (1 mg ml1), were added to the amino-functionalized nanoparticles for the covalent attachment of FITC. After stirring at room temperature for 18–20 h, the particles were purified several times by consecutive ultracentrifugation and redispersion in pure water by ultrasonication as described above to remove unreacted reagents. The final volume after redispersion was 7 ml of water. The concentration of calcium in the nanoparticle dispersion was determined to 82.2 lg ml1 by AAS, corresponding to 206 lg ml1 calcium phosphate (as hydroxyapatite). For covalent attachment of SNARF-1 (carboxylic acid, acetate, succinimidyl ester; Life Technologies), we added 100 ll of SNARF1 DMSO solution (0.5 mg ml1) to 10 ml of the dispersion of CaP/PEI/SiO2/NH2 nanoparticles. Then the mixture was stirred for 18–20 h at room temperature and purified by ultrasonication/redispersion as described above for FITC. We assumed the same nanoparticle concentration as with FITC-conjugated nanoparticles because of the identical synthetic route. 2.3. Characterization
Polyethylenimine-stabilized calcium phosphate nanoparticles (CaP/PEI) were prepared as previously described [30]. Aqueous solutions of calcium lactate (18 mM, pH 10), (NH4)2HPO4 (10.8 mM, pH 10) and PEI (2 g l1; Sigma–Aldrich; branched; 25 kDa) were simultaneously pumped in a volume ratio of 5 ml: 5 ml: 7 ml into a stirred glass vessel containing 20 ml of ultrapure water during 1 min at room temperature. After 20 min stirring, 10 ml of the CaP/PEI-nanoparticle dispersion was added to a mixture of 40 ml ethanol, 50 ll tetraethylorthosilicate (TEOS; Sigma– Aldrich) and 26 ll aqueous ammonia solution (30–33%) for coating with a thin layer of silica. This reaction mixture was stirred for 16 h at room temperature. Then the particles were isolated by ultracentrifugation and redispersed in the original volume of water (10 ml) (UP50H, Hielscher, Ultrasound Technology; sonotrode 7, cycle 0.8,
Scanning electron microscopy was performed with an ESEM Quanta 400 instrument with gold/palladium-sputtered samples. Dynamic light scattering and zeta potential determinations were performed with a Zetasizer nanoseries instrument (Malvern Nano-ZS, laser: k = 532 nm) using the Smoluchowski approximation and taking the data from the Malvern software without further correction. The particle size data refer to scattering intensity distributions (z-average). Ultracentrifugation was performed at 25 °C with an Optima XL-I instrument (Beckman–Coulter). The cells were analysed by flow cytometry with a LSR II instrument using the DIVA software (BD Biosciences). Confocal laser scanning microscopy was performed with a Zeiss LSM 510 Axiovert 200 instrument.
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V. Sokolova et al. / Acta Biomaterialia xxx (2013) xxx–xxx Table 1 Calculated number of dye molecules (CpG and FITC, respectively) per calcium phosphate nanoparticle. Sample
Number of nanoparticles per m3 colloidal dispersion
Number of dye molecules per m3 colloidal dispersion
Number of dye molecules per nanoparticle
CaP/CpG/CaP/CpG CaP/PEI/SiO2/NHFITC
4.75 1016 7.25 1016
6.62 1020 9.76 1021
13,900 135,000
Fig. 2. Schematic representation of the conjugation of SNARF-1 molecules to amino groups on the nanoparticle surface.
2.4. Calculation of the amount of dye per calcium phosphate nanoparticle The nanoparticle concentration was calculated by taking the concentration of calcium phosphate in the dispersion and assuming spherical nanoparticles with a diameter of 120 nm. The number of dye molecules was determined by quantitative UV spectroscopy after previous calibration for CpG-Alexa 555 and FITC, respectively (see Eq. (1) and Table 1):
NðNPÞ ¼
mðCapÞ 3 mðCaPÞ ¼ mðNPÞ 4 p rðNPÞ3 qðCaPÞ
Table 2 Dynamic light scattering (DLS) data of water-dispersed calcium phosphate nanoparticles (PDI: polydispersity index). Sample
Hydrodynamic diameter (nm)
PDI
Zeta potential (mV)
CaP/CpG/CaP/CpG CaP/PEI/SiO2/NHFITC CaP/PEI/SiO2/NHSNARF
322 183
0.348 0.242
25 ± 4 +21 ± 4
195
0.356
+22 ± 5
ð1Þ
NðdyeÞ ¼ cðdyeÞ NA 3
where N(NP) is the number of nanoparticles per m ; m(CaP) is the mass of all synthesized calcium phosphate nanoparticles per m3 with m(CaP/CpG/CaP/CpG) = 0.135 kg m3 and m(CaP/PEI/SiO2/NH-FITC) = 0.206 kg m3, respectively; m(NP) is the mass of one calcium phosphate nanoparticle: m(NP) = 2.84 1018 kg; r(NP) is the average radius of one nanoparticle: r(NP) = 60 109 m; q(CaP) is the density of calcium phosphate: q(CaP as hydroxyapatite) = 3140 kg m3; N(dye) is the number of dye molecules per m3; c(dye) is the concentration of the dye in the nanoparticle dispersion: c(CpG-Alexa 555) = 0.00780 kg m3/(7.1176 kg mol1) = 0.00110 mol m3 and c(FITC) = 0.00631 kg m3/(0.3894 kg mol1) = 0.0162 mol m3, respectively; and NA is the Avogadro constant: NA = 6.022 1023 mol1. 2.5. Nanoparticle uptake HeLa cells (human epithelial cervical cancer cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS) at 37 °C and 10% CO2 and subcultivated according to standard cell culture protocols. 12 h prior to uptake experiments, the cells were trypsinized and seeded in six-well plates with 1 105 cells per well in 2 ml DMEM with FCS. The following final concentrations of calcium phosphate nanoparticles per well
were tested: 2.30 109 ml1, 3.57 109 ml1, 5.17 109 ml1, 6.65 109 ml1 for CaP/CpG/CaP/CpG and 0.072 109 ml1, 0.18 109 ml1, 0.72 109 ml1, 1.77 109 ml1 for CaP/PEI/ SiO2/NH-FITC nanoparticles. The cells were incubated for 3 h, then the whole cell culture medium was removed. The cells were washed three times with phosphate-buffered saline (PBS). Afterwards, the cells were trypsinized, centrifuged and resuspended in 200 ll of FACS solution. The cells were then analysed by flow cytometry. 2.6. Immunofluorescence and localization of nanoparticles in the cell HeLa cells were cultured in DMEM, supplemented with 10% FCS at 37 °C and 10% CO2 and subcultivated according to standard cell culture protocols. 12 h prior to the uptake experiments, the cells were trypsinized and seeded in cell culture dishes with 5 104 cells per well in 0.5 ml DMEM with FCS. The incubation with either cationic or anionic nanoparticles was carried out as follows. Either 40 ll (corresponding to 3.57 109 nanoparticles ml1 after dilution) of CaP/CpG/CaP/CpG nanoparticles or 5 ll (corresponding to 0.72 109 nanoparticles ml1 after dilution) of CaP/PEI/SiO2/NH-SNARF nanoparticles were added, the cells were incubated for 3 h and then the whole cell culture medium was removed. The cells were washed three
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Fig. 3. Scanning electron micrographs of negatively charged CaP/CpG/CaP/CpG nanoparticles and of positively charged CaP/PEI/SiO2/NH-dye nanoparticles (dye = FITC or SNARF), together with a schematic representation of their multi-shell structure.
Fig. 4. Functionalized calcium phosphate nanoparticles are increasingly taken up by cells when their concentration increases. The cells were incubated with different amounts of anionic CaP/CpG/CaP/CpG nanoparticles (labelled with Alexa488; (A)) and cationic CaP/PEI/SiO2/NH-FITC nanoparticles (B) for 3 h at 37 °C. Mock: cells without nanoparticles.
times with PBS to remove adhering nanoparticles. Therefore, only nanoparticles which were either taken up by the cells or strongly adsorbed onto the cellular surface remained. Afterwards, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then washed three times with PBS. Then the cells were stained for 15 min with 1 ll of anti-LAMP-1 staining reagent (mouse anti-human CD107A/LAMP-1 FITC conjugate; Life Technologies) for lysosomes (0.5 lg ml1) and washed three times with PBS. The cells were then stained with DAPI for the cell nucleus
and washed three times with PBS. Finally, the cells were studied by confocal laser scanning microscopy. The cellular uptake kinetics of CaP/PEI/SiO2/NH-SNARF nanoparticles by HeLa cells was studied as follows. The incubation was performed for 3, 5, 13 and 25 h with 5 ll (0.72 109 nanoparticles ml1 after dilution) of CaP/PEI/SiO2/NH-SNARF nanoparticles in cell culture dishes with 5 104 cells per well in 0.5 ml DMEM with FCS. After these times, the whole cell culture medium was removed, and the cells were washed three times with PBS. Then the
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Fig. 5. MTT test of HeLa cells treated with different amounts of anionic CaP/CpG/CaP/CpG nanoparticles (labelled with Alexa488; (A)) and cationic CaP/PEI/SiO2/NH-FITC nanoparticles (B). Mock: cells without nanoparticles.
Fig. 6. FACS data of the cellular uptake of negatively charged calcium phosphate nanoparticles (CaP/CpG/CaP/CpG; labelled with Alexa 488) at 4 °C and in the presence of various inhibitors of endocytosis. Wortmannin and LY294002 efficiently blocked the nanoparticle uptake, indicating macropinocytosis as uptake mechanism.
cells were stained with DAPI (40 ,6-diamidino-2-phenylindole dilactate), washed three times with PBS and studied by confocal laser scanning microscopy.
solution and analysed by flow cytometry. Statistical analysis of the mean fluorescence for each treatment compared to the untreated cells (mock) was done using ANOVA in Sigmaplot.
2.7. Blocking nanoparticle uptake using inhibitors of endocytosis
2.8. Cytotoxicity of nanoparticles
Cells were plated in six-well plates with 105 cells per well in 2 ml DMEM with FCS 1 day before the uptake experiments. The cells were pre-incubated with various inhibitors: wortmannin (100 ng ml1), LY294002 (20 lg ml1), nocodazole (10 lg ml1), chlorpromazine (1 lg ml1) and nystatin (10 lg ml1) for 30 min, followed by the addition of CaP/CpG/CaP/CpG (5.17 109 nanoparticles ml1 after dilution) or CaP/PEI/SiO2/NH-FITC (0.72 109 nanoparticles ml1 after dilution) and incubation for 3 h. The cells were then washed three times with PBS (4 °C). Afterwards, the cells were trypsinized, centrifuged, resuspended in 200 ll of FACS
The cell viability was analysed by an MTT assay 3 h after the incubation with the following concentrations of calcium phosphate nanoparticles per well after dilution: 2.30 109 ml1, 3.57 109 ml1, 5.17 109 ml1, 6.65 109 ml1 for CaP/CpG/CaP/CpG nanoparticles and 0.072 109 ml1, 0.18 109 ml1, 0.72 109 ml1, 1.77 109 ml1 for CaP/PEI/SiO2/NH-FITC nanoparticles under the same conditions as in the uptake studies described above. MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Sigma, Taufkirchen, Germany) was dissolved in PBS (5 mg ml1) and then diluted to 1 mg ml1 in the cell culture medium. The cell culture
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Fig. 7. The uptake of calcium phosphate nanoparticles in the presence of endocytosis inhibitors, normalized to the uptake in the absence of inhibitors (100%). (A) CaP/CpG/ CaP/CpG (anionic; labelled with Alexa488); (B) CaP/PEI/SiO2/NH-FITC (cationic). ( means a significance of P < 0.05).
Fig. 8. Confocal laser scanning microscopy of HeLa cells after 3 h of incubation with negatively charged CaP/CpG/CaP/CpG nanoparticles ((labelled with Alexa555; (I)) and positively charged CaP/PEI/SiO2/NH-SNARF nanoparticles (II). (A) DAPI staining; (B) anti-LAMP-1 staining for lysosomes; (C) red fluorescent nanoparticles; (D) merged image; the yellow colour indicates a colocalization of green anti-LAMP-1 and red fluorescing nanoparticles. Scale bar = 5 lm.
medium of the incubated cells was replaced by 300 ll of the MTT solution and incubated for 1 h at 37 °C under 5% CO2 in humidified atmosphere. 300 ll DMSO were added to the cells. After 30 min, a 100 ll aliquot was taken for spectrophotometric analysis with a Multiscan FC instrument (Thermo Fisher scientific, Vantaa, Finland) at k = 570 nm. The absorption of incubated cells was normalized to that of control (untreated) cells, thereby indicating the relative level of cell viability.
3. Results and discussion Positively charged calcium phosphate nanoparticles were prepared by covalent functionalization as described earlier [30]. FITC was attached by reaction of the amino groups on the particle surface with the isothiocyanate group of FITC [30], giving CaP/PEI/ SiO2/NH-FITC nanoparticles. SNARF-1 was conjugated by a reaction between the amino groups on the particle surface with the succinimidyl-groups of functionalized SNARF-1 molecules (Fig. 2), giving CaP/PEI/SiO2/NH-SNARF nanoparticles. Negatively charged triple-
shell nanoparticles were prepared by multi-step precipitation, giving CaP/CpG/CaP/CpG nanoparticles. All functionalized calcium phosphate nanoparticles were characterized by dynamic light scattering and scanning electron microscopy (Table 2). A spherical morphology of the nanoparticles with a diameter of 120 nm was shown by scanning electron microscopy (SEM; Fig. 3). To follow the uptake of calcium phosphate nanoparticles into cells, we incubated HeLa cells with different concentrations of nanoparticles (2.30–6.65 109 nanoparticles ml1 of CaP/CpG/ CaP/CpG (labelled with Alexa488) and 0.072–1.77 109 nanoparticles ml1 of CaP/PEI/SiO2/NH-FITC). The resulting fluorescence per cell, which is a measure of the amount of uptaken nanoparticles, was determined by flow cytometry. As shown in Fig. 4, a higher concentration of fluorescent calcium phosphate nanoparticles led to an increased uptake and in turn to a larger average fluorescence per cell. The viability of cells treated with increasing amounts of calcium phosphate nanoparticles was assessed by the MTT test. The viability of HeLa cells after incubation with negatively or positively charged nanoparticles for 3 h was 90–100% and did not depend
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Fig. 9. Confocal laser scanning microscopy (CLSM) of HeLa cells after incubation with positively charged CaP/PEI/SiO2/NH-SNARF nanoparticles for 3 h, 5 h, 13 h and 25 h, respectively. (A) DAPI-channel (cell nucleus), (B) FITC-channel (nanoparticles at low pH in lysosomes), (C) TRITC-channel (nanoparticles at neutral pH in endosomes) and (D) merged image. Mock: cells without nanoparticles.
on the concentration (Fig. 5). We conclude that neither kind of nanoparticles was cytotoxic. The route of entry of nanoparticles into the cell and their final intracellular localization is decisive for a potential application as gene or drug delivery agent. We have used several inhibitors of endocytosis to elucidate the mechanism of the cellular uptake of negatively and positively charged calcium phosphate nanoparticles. HeLa cells were incubated for 30 min with the inhibitors and then treated either with CaP/CpG/CaP/CpG (anionic) or with CaP/PEI/SiO2/NH-FITC (cationic) nanoparticles. As a control, we used cells that had been incubated with nanoparticles in the absence of inhibitors. As negative control, the cells were incubated with nanoparticles at 4 °C for 3 h. As endocytosis is an energyconsuming process for the cell, it is generally inhibited at low temperature [12]. A decrease in the measured nanoparticle fluorescence, compared to the control, indicates the involvement of the specific endocytotic mechanism targeted by that inhibitor (Fig. 6). A statistically significant inhibition of the uptake of negatively charged nanoparticles was observed for wortmannin and
LY294002 (Fig. 7A). Therefore, the inhibition of uptake by wortmannin and LY294002 suggests that nanoparticles enter the cell by macropinocytosis [31,32]. A slight decrease in the uptake was induced by nocodazole for CaP/CpG/CaP/CpG nanoparticles. For positively charged calcium phosphate nanoparticles, a significant inhibition of the uptake was found in the case of wortmannin (Fig. 7B). No reduction in uptake with both types of nanoparticles was observed for either chlorpromazine or nystatin, which inhibit clathrin- or caveolin-mediated endocytosis, respectively [33,34]. Together these data show that the uptake of calcium phosphate nanoparticles into HeLa cells occurs by macropinocytosis rather than by clathrin- or caveolin-mediated endocytosis. The localization of nanoparticles was studied by confocal laser scanning microscopy. Endocytosis begins with the formation of membrane vesicles, continues with the transformation into early endosomes and their further maturation into late endosomes before fusion with lysosomes [35]. Lysosomal-associated membrane protein-1 (LAMP-1) was stained to visualize the lysosomes in the
Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034
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cell. In Fig. 8 it is clearly shown that red-fluorescing nanoparticles are localized in lysosomes after 3 h of incubation. The intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport and endocytosis. pH-sensitive fluorophores have therefore been widely applied to monitor changes in the intracellular pH [36]. Among these pH-sensitive dyes, SNARF-1 gives a significant fluorescence emission shift from red to green when the pH is changed from neutral to acidic [37]. The local measurement of the pH after particle uptake sheds light on the localization inside the cell, e.g. within a lysosome with low pH [38–40]. SNARF-1-conjugated calcium phosphate nanoparticles were used for monitoring of the local pH around nanoparticles during their uptake [12]. CaP/PEI/SiO2/NH-SNARF nanoparticles emit red fluorescence at neutral pH whereas nanoparticles internalized by cells emit green fluorescence when they experience the acidic pH within an endosomal/lysosomal compartment [40]. Fig. 9 shows the fluorescence micrographs of HeLa cells after different incubation times with SNARF-1 pH-sensitive conjugated calcium phosphate nanoparticles. After 3 h of incubation, the nanoparticles start to enter lysosomes, as indicated by the color change of the nanoparticles from red to green. This process continues after 5 h and more. Note that in the endosomes/lysosomes, typically both colors (red and green) are located due to the presence of both forms of SNARF-1 at this moderately low pH (4 to 5). Under these conditions, calcium phosphate nanoparticles will be dissolved. This supports earlier reports that excessive concentrations of calcium phosphate (nano-)particles can lead to cell apoptosis due to an increased intracellular calcium level [25–27,41]. However, we have shown earlier that the released calcium is not harmful for the cells if moderate concentrations of calcium phosphate nanoparticles are used [27]. 4. Conclusions Fluorescent cationic and anionic calcium phosphate nanoparticles are increasingly taken up by cells when their concentration increases. The uptake pathway into HeLa cells is mainly macropinocytosis, leading to their localization first in endosomes and then in lysosomes. After 3 h, the nanoparticles have been taken up by cells and will start to dissolve under the acidic conditions in the lysosome. Thus, they are well suited as non-toxic carriers of all kinds of molecules across the cell membrane into the cytoplasm. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Transregio 60) to A.M.W., J.B. and M.E. We thank Prof. Dr. Eric Metzen and Melanie Baumann for help with confocal laser scanning microscopy. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2, 6, 8 and 9, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2013.02.034. References [1] Bausinger R, von Gersdorff K, Braeckmans K, Ogris M, Wagner E, Bräuchle C, et al. The transport of nanosized gene carriers unraveled by live-cell imaging. Angew Chem Int Ed 2006;45:1568–72.
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Please cite this article in press as: Sokolova V et al. Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells. Acta Biomater (2013), http://dx.doi.org/10.1016/j.actbio.2013.02.034