Surface Functionalization of Silica Nanoparticles ... - ACS Publications

Apr 23, 2012 - Department of Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. •S Supporting Informa...
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Surface Functionalization of Silica Nanoparticles Supports Colloidal Stability in Physiological Media and Facilitates Internalization in Cells Christina Graf,*,† Qi Gao,† Irene Schütz,‡ Christelle Njiki Noufele,† Wentao Ruan,† Uta Posselt,† Elena Korotianskiy,† Daniel Nordmeyer,† Fiorenza Rancan,§ Sabrina Hadam,§ Annika Vogt,§ Jürgen Lademann,§ Volker Haucke,‡ and Eckart Rühl*,† †

Physikalische Chemie, Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195 Berlin, Germany Biochemie, Institut für Chemie und Biochemie, Freie Universität Berlin, Takustr. 6, 14195 Berlin, Germany § Department of Dermatology and Allergy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany ‡

S Supporting Information *

ABSTRACT: The influence of the surface functionalization of silica particles on their colloidal stability in physiological media is studied and correlated with their uptake in cells. The surface of 55 ± 2 nm diameter silica particles is functionalized by amino acids or amino- or poly(ethylene glycol) (PEG)terminated alkoxysilanes to adjust the zeta potential from highly negative to positive values in ethanol. A transfer of the particles into water, physiological buffers, and cell culture media reduces the absolute value of the zeta potential and changes the colloidal stability. Particles stabilized by L-arginine, L-lysine, and amino silanes with short alkyl chains are only moderately stable in water and partially in PBS or TRIS buffer, but aggregate in cell culture media. Nonfunctionalized, N-(6-aminohexyl)-3-aminopropyltrimethoxy silane (AHAPS), and PEGfunctionalized particles are stable in all media under study. The high colloidal stability of positively charged AHAPSfunctionalized particles scales with the ionic strength of the media, indicating a mainly electrostatical stabilization. PEGfunctionalized particles show, independently from the ionic strength, no or only minor aggregation due to additional steric stabilization. AHAPS stabilized particles are readily taken up by HeLa cells, likely as the positive zeta potential enhances the association with the negatively charged cell membrane. Positively charged particles stabilized by short alkyl chain aminosilanes adsorb on the cell membrane, but are weakly taken up, since aggregation inhibits their transport. Nonfunctionalized particles are barely taken up and PEG-stabilized particles are not taken up at all into HeLa cells, despite their high colloidal stability. The results indicate that a high colloidal stability of nanoparticles combined with an initial charge-driven adsorption on the cell membrane is essential for efficient cellular uptake.

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INTRODUCTION Nanoparticles can be prepared with variable size, shape, and surface functionalization, which allow for the adjustment of their physical and chemical properties over a broad range. Due to their unique properties, they serve as fundamental building blocks for various applications in technology,1 pharmaceutics,2−4 and medical applications, such as in vivo targeting of cancer cells, and they are also used as contrast agents and diagnosis.4−6 Progress in these fields implies an extended use of such intentionally made nanoparticles. This involves extensive release of such nanoparticles into the environment. At the present time, the risks of hazards caused by nanoparticles to organisms are still not fully known. This is specifically the case for the uptake by plants, animals, and human beings.7−9 Therefore, investigations on possible hazardous effects related to the exposure of organisms to nanomaterials have recently become an important field of research.9,10 This includes © 2012 American Chemical Society

systematic research on intentionally made nanoparticles and their transport routes into cells and organisms. The initial step of interaction between nanoparticles and cells is particle adsorption on the cell membrane, which is followed by uptake through the membrane into the interior of the cell.11 The most important uptake pathways for submicrometer particles are energy-dependent non-phagocytic routes,12,13 including clathrin-mediated endocytosis14,15 and caveolar uptake.16 Additional, less well understood clathrin- and caveolin-independent routes also exist.17 These include, e.g., macropinocytosis, which allows the uptake of particles and aggregates (up to 500 nm diameter) from the extracellular space.18 Which of these pathways is taken depends on the nature of the nanoparticles and on the cell type. Cell membrane Received: December 14, 2011 Revised: April 22, 2012 Published: April 23, 2012 7598

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Figure 1. TEM images of (a) nonfunctionalized, (b) APS-functionalized, and (c) AHAPS-functionalized silica particles in ethanol. The insets show the same particles in higher magnification.

lished.38−40 and a wide variety of coating procedures using silica has been developed.32−35,41 Besides reducing cytotoxicity, silica shells also allow one to stabilize nanocrystals, such as palladium particles,42 and can improve the photoluminescent stability of fluorescent sensors and biomarkers.43−45 Pure silica particles are often used as model systems in biomedical studies, 46,47 since they can be prepared with a low polydispersity (5000 g/mol) can enhance protein adsorption and, hence, cellular uptake.100 By contrast, the molar mass of the PEG used in the present study is significantly lower, i.e., in the range of 450−580 g/mol, explaining qualitatively the difference in uptake properties. We also note that the cellular uptake of silica particles coated by PEG ligands, similar to those used in the present work, has not been studied before. In the case of APS-functionalized particles, large perinuclear aggregates are observed in confocal spinning disk microscopy images. This suggests that APS particles undergo efficient adsorption to the cell membrane but display a limited ability to subsequently become internalized into HeLa cells. This may be caused by the propensity of these particles to aggregate upon transfer into cell culture media (DMEM + 10% FCS). To ensure that at least some uptake of APS particles occurs, we reconstructed HeLa cells incubated with APS-functionalized nanoparticles for 4 h in 3D. The data clearly show that at least a fraction of the particles adsorbed to the cell surface becomes internalized and accumulates in the perinuclear area of the cell (see Figure S2 and Movie S1 in the Supporting Information). Evidently, cellular uptake/surface binding of the APS particles is more efficient than colloidal aggregation. The APS particles have a highly positive zeta potential in ethanol due to their amino functionalization (+64 ± 3 mV, see Table 1), which decreases as a result of the continuous loss of amino groups from the particle surface after their transfer into aqueous media (see above), where the average zeta potential in water during the first hour is +16 ± 2 mV, and an accurate measurement in DMEM ± 10% FCS is not possible due to severe aggregation and partial precipitation of the particles. Therefore, the improved cellular binding and uptake of APS-functionalized particles is rationalized by their high positive zeta potential, which promotes their electrostatic interaction with the cell membrane. In contrast to APS-functionalized particles, Nguanylpyrazole (GP)-functionalized particles, which also form large aggregates and partially precipitate in DMEM + 10% FCS (see above), are not taken up by HeLa cells. Here, a thick layer of aggregates is found on the cell surface (see Figure 8e), but no labeled particles are detectable in the cytosol or in transferrin-positive endosomes. These data support the observation that their highly positive surface charge paired with favorable colloidal stability enables APS-functionalized silica nanoparticles to be endocytosed efficiently into HeLa cells via mechanism(s) that remain to be determined.

Article

CONCLUSIONS

Nearly monodisperse (55 ± 2 nm diameter) silica nanoparticles with a fluoresceine isothiocyanate (FITC)-labeled core are prepared. Surface functionalization by various amino-terminated alkoxysilanes, amino acids, as well as poly(ethylene glycol)-terminated alkoxysilanes allows the adjustment of the zeta potential of these nanospheres from highly negative to positive values (−56 ± 2 mV to +64 ± 3 mV) in ethanol. A transfer of these nanoparticles into water, physiological buffers, such as phosphate buffered saline (PBS) and TRIS, as well as cell culture media (DMEM, DMEM with 10% FCS, and RPMI) results in a reduction of the zeta potential and changes in colloidal stability. These changes depend strongly on surface functionalization. Silica particles stabilized by L-arginine, Llysine, and aminosilanes with short alkyl chains, such as (3aminopropyl)-trimethoxysiliane (APS) or N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (NPC), are only fairly stable in water and partially stable in PBS or TRIS. Large aggregates are formed or precipitation occurs in the other media. The aggregation kinetics of APS functionalized particles in water follows an exponential growth, which is a consequence of the decrease in electrostatic stabilization. This is due to loss of APS from the silica particle surface, which is promoted by formation of a stable cyclic intermediate. Only nonfunctionalized particles, N-(6-aminohexyl)-3-aminopropyltrimethoxy-silane (AHAPS), and 2-[methoxy(polyethleneoxy)propyl]trimethoxy-silane (PEG)-functionalized particles are reasonably stable in all media under study. The negatively charged nonfunctionalized silica particles show a slow time-dependent aggregation process in water, PBS, DMEM, and RPMI, reaching a constant, slightly increased diameter after several hours. Their colloidal stability in DMEM and RPMI is rather low, where almost a doubled hydrodynamic diameter is reached 14 h after transfer. Their colloidal stability in these cell culture media is significantly lower than in NaCl solution of similar ionic strength, which is rationalized by the strong adsorption of divalent Ca2+ and Mg2+ ions from the media on the particle surface, leading to a partially positive surface charge.92,99 Note that the applied models fitting the experimental results cannot be generalized for a broad range of colloidal systems, but they rather allow a detailed local description of aggregation in the regime of reaction-limited colloidal aggregation. The stability of the nonfunctionalized silica particles significantly increases when proteins, such as those found in 10% fetal calf serum (FCS), are added. Details on the adsorption pattern and amount of proteins adsorbed on the nanoparticles with different functionalizations will be studied in subsequent work. Positively charged AHAPS-functionalized silica particles show weak, time-dependent aggregation in water, PBS, and DMEM, also reaching a constant hydrodynamic diameter after several hours, whereas the aggregation process is nearly time-independent in TRIS and RPMI. In general, their colloidal stability is fairly high in all media and scales with the ionic strength of the solvents, which indicates that electrostatic stabilization dominates and chemical reactions with components of the media are of minor importance. Detachment of their significantly longer amino-alkyl chains from the silica surface does not take place, since a stable cyclic intermediate cannot be formed, as observed for APS-functionalized particles. The PEG-functionalized particles show no or only minor aggregation in cell culture media and physiological buffers, which is not associated with an increase in the ionic strength, 7609

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trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (NPC), (3-aminopropyl)trimethoxysilane (APS), N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS), APS subsequently modified with 1-H-pyrazole-1-carboxamidine hydrochloride (N-guanylpyrazole, GP) or polyethylene functionalization using covalently bound 2-[methoxy(polyethleneoxy)propyl]trimethoxy-silane (PEG-silane) are given in the Supporting Information. Subsequently, all particles are purified by repeated centrifugation and redispersion in ethanol. The particles are stored in ethanol at +4 °C under argon in the dark. Transfer of the Functionalized Silica Nanoparticles from Ethanol to Physiological Media. The transfer process is carried out under argon and in the dark. The ethanol in the dispersion of functionalized silica particles is removed by centrifugation (2800 g, 45 min, T = 20 °C). 50 mL centrifuge tubes (SuperClear PP, VWR collection) are used for all centrifugation steps. The sediment containing the functionalized silica nanoparticles and some of the remaining liquid is redispersed in water using ultrasonication for 20 min (Bandelin Sonorex, RK 512 H 860 W). This procedure is repeated at least twice to ensure that the ethanol content in the samples is negligible (≪0.5 vol %) and no ethanol-related cytotoxicity might be expected. After the last centrifugation step, the particles are redispersed in the physiological medium also using ultrasonication for 20 min (Bandelin Sonorex, RK 512 H 860 W). Characterization of the Functionalized Silica Nanoparticles. Zeta potential and size distribution measurements are performed using a Delsa Nano C from Beckman Coulter. In all DLS measurements, the scattering angle is fixed at 165°. The samples are measured after transferring them into water-free ethanol, highly pure water (Milli-Q) with a resistance of 18.2 MΩ•cm, standard cell culture media (RPMI, DMEM, and DMEM with 10% FCS), buffer solutions (PBS and TRIS), or NaCl solutions. All samples are filtered by a RC syringe filter of 0.2 μm pore size (Carl Roth) before use. The particle concentration is 0.5 g/L for all DLS and zeta potential measurements. The concentrations of the dispersions are determined by drying a defined volume and weighing the remaining residue. TEM images are recorded using an EM 902A TEM from Philips. The TEM samples are prepared by dipping 400 mesh copper grids coated by a ∼15 nm carbon film (Quantifoil) into a dispersion of the nanoparticle samples. The average diameter and the polydispersity of the nanoparticles are analyzed by using the software Simple PCI from C-Images. Cell Cultures. HeLa cells are cultured in low-glucose DMEM supplemented with 10% FCS and antibiotics in a humidified incubator at 37 °C and 5% CO2. Nanoparticle Uptake Followed by Transferrin Uptake. Cells are seeded on poly(L-lysine)-coated glass coverslips and grown to a confluence of 60−70%. After one washing step with PBS, the cells are incubated for 4 h with nanoparticles (100 μg/mL) in full medium in a humidified incubator at 37 °C and 5% CO2. Afterward, incubation with Alexa 568-transferrin (20 μg/mL) is performed for 15 min at 37 °C. Subsequently, the cells are placed on ice, washed three times for 5 min with ice-cold PBS buffer containing 10 mM MgCl2 and fixed by 4% paraformaldehyde, 4% sucrose in PBS, pH 7.4, for 20 min. After fixation, the cells are washed twice in PBS and they are mounted onto glass slides using Immumount solution (Thermo Scientific) supplemented with 1 μg/mL DAPI. Confocal Fluorescence Microscopy. After mounting, HeLa cells are imaged using a Zeiss Axiovert 200M-based PerkinElmer Life Sciences UltraView ERS dual spinning-disk system. 3D reconstruction is processed using Volocity software (Improvision).

even if their zeta potential is reduced. This behavior is explained by an additional steric stabilization by the PEG chains. The fact that time-dependent aggregation occurs already in standard physiological media, which are normally used in cell experiments, indicates that transfer and purification processes of nanoparticles can largely influence their interaction with cells. Severe aggregation may occur even if the particles have a high zeta potential and a high colloidal stability in other polar media. This is particularly true for cell culture media of complex composition, which may efficiently change the colloidal stability of nanoparticles. The strong time dependence of these processes indicates that detailed transfer protocols are required for particle uptake studies. AHAPS-functionalized nanoparticles are efficiently taken up into HeLa cells in DMEM + 10% FCS, since their positive zeta potential enhances the interaction of the nanoparticles with the negatively charged cell membrane and their small hydrodynamic diameter is compatible with endocytic uptake. GPfunctionalized particles also have a positive zeta potential and efficiently attach to the cell membrane. However, no uptake is observed, which is likely due to severe aggregation. Also, positively charged APS particles are internalized into HeLa cells, but due to their time-dependent strongly decreasing surface charge and loss of colloidal stability, this process is less efficient than for AHAPS particles. Negatively charged nonfunctionalized particles, in spite of the fact that they do not form large aggregates, display a very low propensity for internalization into HeLa cells. This is consistent with the view that an initial charge-driven adsorption onto the cell membrane is essential for effective uptake. In the case of PEGfunctionalized particles, which present the highest stability of all samples in DMEM + 10% FCS, no uptake into HeLa cells is observed as PEG functionalization interferes with adsorption onto cell membranes. These results indicate that efficient cellular uptake is based on two major prerequisites: (i) high colloidal stability in cell culture media and (ii) a highly positive zeta potential. The studies further emphasize the importance of quality control not only during the production process of particles, but also during experiments. Secondary effects caused by experimental conditions, such as exposure to cell culture media, may be important confounding factors in experiments which involve particles and biological systems. Further, a negative zeta potential and an effective functionalization with PEG can effectively reduce the uptake of nanoparticles into cells. TEM studies will be carried out in future work to further understand the mechanisms for cellular uptake of nanoparticles as a function of their surface functionalization.

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METHODS AND MATERIALS

All chemicals were purchased from Sigma-Aldrich, Alfa Aesar, or ABCR and are used without further purification. Standard cell culture media (DMEM and RPMI), FCS, and buffer solutions (PBS and TRIS) were received from Roth, Lonza, and Gibco. A detailed list of all chemicals used in the work is given in the Supporting Information. Synthesis of FITC-Dye Labeled Monodisperse Silica Nanoparticles. FITC-dye labeled silica nanoparticles cores with a low polydispersity (diameter of 50 ± 2 nm) are prepared by a modified microemulsion synthesis.56 Details are given in the Supporting Information. Subsequently, an unlabeled silica shell is grown on the silica cores by a modified Stöber process.52,59−61 Functionalization of FITC Labeled Core/Shell Silica Nanoparticles. Details on the surface functionalization of the nanoparticles by adsorbing L-lysine and L-arginine or covalently binding N-

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ASSOCIATED CONTENT

S Supporting Information *

A list of all used chemicals including a table with the exact composition of the cell culture media RPMI 1640 and DMEM, as well as a detailed description of the synthesis of FITC labeled silica nanoparticles and their functionalization are given. Additional data on the colloidal stability of AHAPS and PEGfunctionalized as well as nonfunctionalized silica particles in 7610

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water, physiological buffers, and cell culture media are presented. Further, data on the colloidal stability of the nanoparticles as a function of the ionic strength of the NaCl solution are shown. A 3D-reconstruction of HeLa cells after a 4 h incubation with APS-functionalized nanoparticles and a movie of a 3D-reconstruction of two HeLa cells after a 4 h incubation with APS-functionalized nanoparticles and 15 min transferrin uptake are presented as well. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*C. Graf: Tel. +49 30 83855304, Fax. +49 30 83852717, E-mail [email protected]. E. Rühl: Tel. +49 30 83852396, Fax. +49 30 83852717, E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (SPP 1313, Cluster NanoSelect (RU 420/9-1, RU 420/ 9-2, HA2686/5-1; SFB 765/B4 and C5) and the Freie Universität Berlin. We thank Dr. H. Renz and Prof. Dr. R. J. Radlanski (Department of Craniofacial Developmental Biology, Charité - Universitätsmedizin Berlin) for the use of their electron microscope.

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