Surface charge effect on the cellular interaction and cytotoxicity of NaYF₄:Yb³⁺, Er³⁺@SiO₂ nanoparticles

Abstract

Taking advantage of the unique optical properties of upconversion nanoparticles (NP) and the feasibly modified nature of SiO₂, NaYF₄:Yb³⁺, Er³⁺@SiO₂ core@shell (UCNP@SiO₂) NPs with excellent morphology have been synthesized to investigate the surface charge effect on the interactions of NPs with cells. The cellular uptake behaviors, cytotoxicities and intracellular metal ion release of the as-prepared NPs have been investigated in two cell lines (HeLa and HepG-2) by fluorescence microscopy, MTT assay and inductively coupled plasma mass spectrometry (ICP-MS), respectively. It is found that the UCNP@SiO₂ NPs with positive surface charge have much higher cell internalization ability and cytotoxicity than those with negative surface charge. The experimental results also demonstrate that the cytotoxicity of UCNP@SiO₂ NP is closely related to the increase of intracellular reactive oxygen species (ROS) and metal ion release of internalized NPs.

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Introduction

Functionalized and/or engineered nanoparticles (NPs) have been extensively employed as basic units to construct novel compounds/devices for many different fields ranging from clean energy to healthcare.^1–10 However, there is a controversy surrounding the biological safety of nanomaterials which seriously hampers the translation of nanotechnological improvements into the field of biomedicine.^11–27 Numerous factors, such as size, shape, surface charge and ligands have been associated with NP cytotoxicity and cellular responses.^11–22 In addition, the elemental toxicity of NP composition can play a role in the case of heavy metal-containing nanoparticles because free metal ions could be released from NP surface in oxidative and/or acidic conditions.²³ The amount of toxicological data produced is steadily increasing by studying the interactions of the wide variety of physicochemical properties of the NPs with the large range of different cell types.^24,25 In general, cytotoxicity is dependent on the NP uptake rate and pathway of internalization since it takes place in the intracellular space. Many studies demonstrated that surface charge plays an important role in the interactions of NPs with cells.^12,14,28 For example, Iyer et al. have demonstrated that quantum dots (QDs) elicited cytotoxicity is a function of a combination of factors where surface charge > surface functionalization (ligand) > NP size.²⁸ In addition, NP's cytotoxicity is also closely related to the tested cell types because of different uptake behaviors of NPs for different cells.^14,29,30 For instance, Ji et al. have demonstrated that the cytotoxicity of gold NPs is quite different between nonphagocytic cells (HepG-2) and phagocytic cells (RAW 264.7).²⁹

Compared to downconversion materials, UCNPs have many advantages including minimum photodamage to living cells, low autofluorescence, high signal-to-noise ratio and detection sensitivity, and high penetration depth in biological or environmental samples, which make the UCNPs particularly useful for bioimaging.^4,31 Recently, silica coated lanthanide-doped upconversion NPs (UCNP@SiO₂) have been given a lot of attention in the development of new generation of imaging agents for bioimaging.^32–37 The wide interest in UCNP@SiO₂ stems from a broad variety of enticing properties that UCNPs (core) and SiO₂ (shell) possess, including: (1) high efficiency and multicolor emissions, (2) monodispersed size and uniform shape, (3) nanochemically engineered surface for phase transfer and for coupling to targeting ligands, and (4) the high biocompatibility. The results of MTT (methyl thiazolyl tetrazolium), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, sodium salts), and CCK-8 mitochondrial metabolic activity assays have suggested that UCNP@SiO₂ are noncytotoxic (cellular viabilities > 90%) to a broad range of tested cell lines within a certain range of concentrations and a limited incubation period.^35,38,39 However, the precise behaviours underlying cytotoxicity of UCNP@SiO₂ are still not completely understood. In particular, the effect of surface charge on cytotoxicity of UCNP@SiO₂ should be carefully studied since the cellular internalization of NPs is strongly dependent on the surface charge.¹⁵

Here, we systemically study the impact of surface charge of UCNP@SiO₂ on the interactions of cells with UCNP@SiO₂ using both HeLa and HepG-2 cells. The 37 nm carboxy-terminated UCNP@SiO₂ with negative surface charge are conjugated with positively charged NH₂-PEG (UCNP@SiO₂-PEG) or NH₂-PEG-NH₂ (UCNP@SiO₂-PEG-NH₂) for generating passivate UCNP@SiO₂ with different surface charges. The uptake of the all NPs into two cells lines was studied by fluorescence microscopy and ICP-MS. The results of MTT and ROS experiments indicate that the cytotoxicity of UCNP@SiO₂ are affected by the NP's surface charge.

Experimental section

Materials

Yttrium oxide (Y₂O₃, 99.99%), ytterbium oxide (Yb₂O₃, 99.99%), and erbium oxide (Er₂O₃, 99.99%) were bought from Alfa Aesar (Ward Hill, USA). YCl₃, YbCl₃ and ErCl₃ aqueous stock solutions were obtained by dissolving corresponding lanthanide oxides in hydrochloric acid, respectively. 1-Octadecene (ODE, 90%), oleic acid (OA, 90%), tetraethyl orthosilicate (TEOS, 99.999%), polyoxyethylene (5) nonylphenyl ether (Igepal CO-520, M_n = 441), ammonia solution (28%) and poly(ethylene glycol)bis(3-aminopropyl) terminated (NH₂-PEG-NH₂, M_n = 1500) were purchased from Sigma-Aldrich Co. (St Louis, USA). Carboxyethylsilanetriol (CTES, sodium salt, 25% in water) was obtained from Gelest Inc. (Morrisville, USA). N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS) and methoxypolyethylene glycol amine (NH₂-PEG, M_n = 2000) were purchased from Aladdin Reagent Co. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beijing Dingguo Biotechnology Ltd. (Beijing, China). Reactive oxygen species (ROS) detection kit was purchased from Beyotime Company (Jiangsu, China). Other reagents (analytical grade) were purchased from Beijing Chemical Reagents Company (Beijing, China). All these reagents were used as received without further purification. Milli-Q water (18.2 MΩ cm) was used for all experiments.

Characterization

Transmission electron microscope (TEM) micrographs were performed with a JEM 2000FX (Jeol Ltd., Japan). Powder X-ray diffraction (XRD) analysis was carried out on a D8 advance diffractometer (Bruker Co., Germany) with Cu Kα (0.15406 nm) radiation. Energy-dispersive X-ray spectra (EDS) were carried out on a field emission scanning electron microscope (FESEM, S4800, Hitachi Co., Japan) equipped with an EDS (Jeol JXA-840). Dynamic light scattering (DLS) and zeta potential measurements were carried out on Malvern zetasizer Nano ZS (Malvern Instruments Ltd., UK). Upconversion luminescence (UCL) spectra were recorded from an F-4500 spectrophotometer (Hitachi Co., Japan) using an external CW NIR laser as excitation light source.

Synthesis of NaYF₄:Yb³⁺, Er³⁺ upconverting NPs

OA-capped NaYF₄:Yb³⁺, Er³⁺ UCNPs were prepared by a previously reported method with slight modifications.³⁴ Typically, 1 mL aqueous solution of LnCl₃ (1 M, Ln = Y, Yb, Er) was added to a 100 mL flask, and dried by heating. OA (6 mL) and 1-octadecene (15 mL) were then added to the flask and the reaction mixture was incubated at 150 °C under Ar atmosphere for 5 min. Following cooled to room temperature, 10 mL methanol solution containing NaOH (2.5 mmol) and NH₄F (4 mmol) was added and vigorously stirred for 60 min. For evaporating the methanol, the solution was heated to 70 °C and degassed at 110 °C for 10 min. Subsequently, the reaction mixture was incubated at 300 °C for 60 min and then cooled down to room temperature. The product was precipitated and washed by addition of 15 mL ethanol (3 times). Finally, as-prepared OA-capped NaYF₄:Yb³⁺, Er³⁺ (UCNPs) were collected by centrifugation and redispersed in cyclohexane. In the following experiments, the concentration of NP was defined by the amount of yttrium (Y) element.

Synthesis of carboxy-functionalized UCNP@SiO₂ core@shell NPs

The carboxy-functionalized SiO₂, UCNP@SiO₂ core@shell NPs (UCNP@SiO₂-COOH) were synthesized by our previously reported strategy.³⁴ Generally, 0.1 mL Igepal CO-520, 8.6 mg OA-capped UCNPs and 10 mL cyclohexane were mixed and stirred for 10 min. 0.4 mL Igepal CO-520 and 0.08 mL ammonium hydroxide (28 wt% in water) were then added into the mixture and sonicated for 20 min. After sonicating, 0.05 mL TEOS was added dropwise and stirred for 5.75 h. Subsequently, 0.02 mL CTES was added into the solution and continually stirred for 2 days. The UCNP@SiO₂-COOH were precipitated, washed and collected as previously described. The as-prepared UCNP@SiO₂-COOH were redispersed in water and stored at room temperature.

Synthesis of amino-PEG-functionalized UCNP@SiO₂ core@shell NPs

0.5 mL EDC (2.5 mg mL⁻¹) and 0.5 mL sulfo-NHS (10 mg mL⁻¹) were mixed with 1 mL UCNP@SiO₂-COOH (1 mg mL⁻¹) in MES buffer (0.1 M, pH 6.0). After gently stirred for 1 h at room temperature, NPs were centrifuged and washed with 2 mL MES buffer (0.1 M, pH 6.0). Subsequently, the NPs were redispersed in 5 mL HEPES buffer solution (0.01 M, pH 7.2) containing 7 mg NH₂-PEG-NH₂. The reaction mixture was incubated for 3 h at 37 °C with reciprocating oscillation (150 rpm). The amino-PEG-functionalized UCNP@SiO₂ core@shell NPs (UCNP@SiO₂-PEG-NH₂) were purified by centrifugation (10 000 rpm, 10 min, 3 times). The as-prepared UCNP@SiO₂-PEG-NH₂ were redispersed in water and stored at room temperature.

Synthesis of PEG-functionalized UCNP@SiO₂ core@shell NPs

The PEG-functionalized UCNP@SiO₂ core@shell NPs (UCNP@SiO₂-PEG) were synthesized by the same synthesis procedure of UCNP@SiO₂-PEG-NH₂, except for the use of NH₂-PEG.

Measurements of cellular internalization of NPs

UCL imaging. HeLa and HepG-2 cells (1 × 10⁵ cell per mL in 1 mL fresh culture medium) were seeded in 12 well cell-culture plate at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h, respectively. Then, the culture medium was discharged and the cells were washed with 1 mL PBS and fresh culture medium, respectively. Subsequently, cells were incubated in 1 mL fresh culture medium containing 25 μg mL⁻¹ for another 24 h at 37 °C under 5% CO₂. After incubating with NPs, the cells were washed with 1 mL fresh culture medium (3 times) and PBS (3 times) to remove any excess NPs. Finally, the UCL images of HeLa and HepG-2 cells were recorded on a reconstructive Nikon Ti–S fluorescent microscope under the excitation of CW 980 nm laser (BWT Beijing Ltd., China).

Measurements of the intracellular amounts of NPs

After treated with 25 μg mL⁻¹ NPs as described above, the NPs stained cells were washed with 1 mL fresh culture medium (3 times) and PBS (3 times), detached from the culture plates by 0.2 mL trypsin, concentrated by centrifugation (500 g), freeze-dried, redispersed with concentrated nitric acid and analyzed the intracellular yttrium content by ICP-MS.

Measurements of intracellular metal ions release of internalized NPs

The HeLa and HepG-2 cells were incubated with 25 μg mL⁻¹ NPs, washed, detached and concentrated as previously described. Then, the NPs stained cells were resuspended in 1 mL lysate buffer (Beijing Dingguo Biotechnology Co., Ltd., China) and incubated at 0 °C for 15 min. The homogenates were centrifuged at 19 000 rpm for 20 min using an H-2050R centrifuge (Xiang Yi centrifuge instrument Co., Ltd., China) at 4 °C. Finally, the supernatants (cell lysates) were collected and analyzed by ICP-MS.

Cytotoxicity measurement

The in vitro cytotoxicities of NPs were studied by traditional MTT assay.⁴⁰ Briefly, HeLa and HepG-2 cells (1 × 10⁴ cells per well) were seeded in 96 well cell-culture plate and cultured at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h. Then, the culture medium was discharged and the cells were washed with 100 μL fresh culture medium and PBS, respectively. Subsequently, the NPs with desired concentrations (0, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 μg mL⁻¹) in 100 μL fresh culture medium were added into the wells and incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 24 h, respectively. After washed as previously described, the cells were treated by MTT (5 mg mL⁻¹ in 100 μL culture medium) and cultured at 37 °C in a humidified atmosphere with 5% CO₂ for another 4 h. After discharged the supernatant, dimethyl sulfoxide (DMSO) were added into the wells (100 μL per well) and shaken for 10 min. Finally, the absorbance (λ = 510 nm) of each well was recorded on a microplate reader (BioTek Instrument Co., Ltd., USA). The relative cell viabilities (%) were calculated by using the optical densities contrast to the control value. In this case, HeLa and HepG-2 cells cultured without NPs were used as control samples.

Measurements of intracellular reactive oxygen species (ROS)

The intracellular level of ROS was monitored by oxidation-sensitive fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)-based assay kit with manufacturer's instructions.⁴¹ Briefly, HeLa and HepG-2 cells were treated with 25 μg mL⁻¹ NPs for 24 h as previously described. The NPs stained cells were washed as previously described, incubated with 10 μmol mL⁻¹ DCFH-DA in 1 mL serum-free medium (DMEM) at 37 °C for 20 min, and washed with 1 mL PBS (pH 7.4, sterilized, 3 times). Then, the cells were detached by trypsin, collected and analyzed by Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc., USA) under an excitation wavelength of 488 nm and emission wavelength of 525 nm.

Results and discussion

Synthesis and characterization of UCNP@SiO₂ core@shell NPs

The UCNP@SiO₂-COOH were synthesized by previously reported water-in-oil microemulsion strategy.³⁴ Generally, the hydrophobic OA-capped UCNP cores (Y : Yb : Er = 80% : 18% : 2%) were reacted with TEOS and CTES in the water-in-oil microemulsion. In particular, CTES was added to the reaction mixture following the addition of TEOS. The UCNP@SiO₂-COOH were generated through the reactions of CTES with silanol groups on TEOS formed silica shell. We found that the morphologies of UCNP@SiO₂-COOH were critically related to the reaction temperature and stirring rate (as shown in Fig. S1 in the ESI†). UCNP@SiO₂-COOH with excellent morphology were obtained at 18 °C and 300 rpm. The TEM images show that the particles have uniform size and shape. In the following studies, the UCNP@SiO₂-COOH were synthesized under this condition. The average size of UCNP@SiO₂-COOH is about 37 nm in diameter, which is composed of a 18 nm UCNP core and a 9.5 nm silica shell (as shown in Fig. 1a and b). The zeta potential (−38.3 mV) of UCNP@SiO₂-COOH suggests that the NPs have strongly negative surface charge (as shown in Table 1). In addition, the corresponding XRD pattern (as shown in Fig. S2 in the ESI†) of the sample shows that the UCNP cores are composed of pure hexagonal phase (Standard PDF card: 16-0334). The elemental composition of the UCNP@SiO₂-COOH is further confirmed by XPS and EDS analysis (as shown in Fig. S3 and S4 in the ESI†).

Fig. 1 TEM images of (a) OA-capped UCNPs, (b) UCNP@SiO₂-COOH, (c) UCNP@SiO₂-PEG and (d) UCNP@SiO₂-PEG-NH₂.

Table 1 UCNP@SiO₂ core@shell NP physicochemical properties^a

	Physical diameter [nm]	Hydrodynamic diameter^a [nm]	Hydrodynamic diameter^b [nm]	Zeta potential^a [mV]	Zeta potential^b [mV]
a The NPs were redispersed in water^a and culture medium^b (DMEM + 10% FBS), respectively. The average physical diameters of NPs are obtained by TEM measurements and the hydrodynamic diameters of NPs are generated by DLS measurements.
UCNP@SiO2-COOH	36.9 ± 1.6	104.7 ± 0.1	121.8 ± 1.1	−38.3 ± 0.5	−10.1 ± 1.3
UCNP@SiO2-PEG	37.8 ± 1.6	146.3 ± 0.6	238.5 ± 4.6	−13.7 ± 0.7	−10.2 ± 0.8
UCNP@SiO2-PEG-NH2	37.7 ± 1.9	139.9 ± 0.6	149.7 ± 5.0	26.2 ± 0.5	−6.3 ± 0.2

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In order to adjust the surface charges of NPs, the as-prepared UCNP@SiO₂-COOH were reacted with NH₂-PEG and NH₂-PEG-NH₂, respectively. The NH₂-PEG and NH₂-PEG-NH₂ were used in the study because the polyethylene glycol derivatives have good biocompativity.^42,43 The FITR spectra of UCNP@SiO₂-COOH, UCNP@SiO₂-PEG and UCNP@SiO₂-PEG-NH₂ are shown in Fig. 2. The increasing of absorption at 1633 cm⁻¹ and 1637 cm⁻¹ (stretching vibration of C=O in amide I bands), 1548 cm⁻¹ (stretching vibration of C=O in amide II bands) and decreasing of absorption at 1417 cm⁻¹ and 1569 cm⁻¹ (asymmetric and symmetric stretching vibration bands of carboxy group) demonstrate that UCNP@SiO₂-PEG and UCNP@SiO₂-PEG-NH₂ have been successfully synthesized. The TEM micrographs of the UCNP@SiO₂-PEG and UCNP@SiO₂-PEG-NH₂ are shown in Fig. 1c and d. The TEM result indicates that the morphology and physical diameter of NPs are not affected by the polyethylene glycol derivative modification. However, the hydrodynamic diameters and zeta potentials of UCNP@SiO₂-PEG and UCNP@SiO₂-PEG-NH₂ are changed significantly by the PEG derivative modification (as shown in Table 1). The hydrodynamic diameters of all NPs in culture medium are larger than their original sizes in water. The experimental result suggests that some serum proteins (e.g., serum albumin) adsorb on the NP surfaces in the culture medium. In the culture medium, the hydrodynamic diameter of UCNP@SiO₂-PEG-NH₂ is smaller than that of UCNP@SiO₂-PEG. The phenomenon may due to that different PEG derivatives modified UCNP@SiO₂ might adsorb different proteins in the cell culture media. Before and after incubation in culture medium, the zeta potential of UCNP@SiO₂-PEG-NH₂ change from positive (26.2 mV) to negative (−6.3 mV), while the zeta potential of UCNP@SiO₂-COOH and UCNP@SiO₂-PEG change to less negative. Similar phenomenon also has been observed of other NPs.¹⁷ The different zeta-potential changes of different NPs could be caused by different protein adsorbing behaviors.^15,44,45 For instance, NPs with positive surface charge prefer to absorb negative proteins while NPs with negative surface charge like absorbing positive proteins. The differences in protein adsorption of NPs could result in the difference between endocytosis.¹⁵ In addition, the UCL spectra of NPs were not changed significantly after modification (as shown in Fig. S5 in the ESI†). In addition, the zeta potential and colloidal stability of UCNP@SiO₂-PEG-NH₂ is much higher than that of 3-aminopropyltrimethoxysilane (APS) modified UCNP@SiO₂ (7.4 mV). And the UCNP@SiO₂-PEG-NH₂ and UCNP@SiO₂-COOH have closed surface charge density (absolute value). Therefore, UCNP@SiO₂-PEG-NH₂ was used to study the effect of positive charge on the cytotoxicity of UCNP@SiO₂.

Fig. 2 FTIR spectra of (a) UCNP@SiO₂-COOH, (b) UCNP@SiO₂-PEG and (c) UCNP@SiO₂-PEG-NH₂, respectively.

Cellular internalization of NPs

Due to the properties of upconversion luminescence, UCNP@SiO₂ can be directly used as fluorescent probes for biological imaging. To investigate the cell uptake behavior of UCNP@SiO₂ affected with different surface charges, the NPs were incubated in HepG-2 cells (human hepatoma cell line, as model cell of detoxification organ) and HeLa cells (cervix epithelioid carcinoma cell line, as model cell of non-detoxification organ), respectively. The different NPs were incubated with cells in the same experimental conditions. The NPs stained cells were observed by UCL imaging and cellular internalized NPs were quantified by ICP-MS with described as Y content in cells. It is clearly observed that the UCNP@SiO₂-NH₂ stained cells have the brightest UCL and UCL intensity of UCNP@SiO₂-COOH stained cells is much stronger than that of UCNP@SiO₂-PEG stained cells (as shown in Fig. 3). The UCL imaging of the cells provides direct evidence on that the NPs are uptaken by cells. To further quantitatively analyze the cellular uptaken amount of the NPs, the NPs stained cells are examined by ICP-MS measurements (as shown in Fig. 4). The result of ICP-MS measurement is consistent with that of UCL imaging experiment. For the same cell line, the uptake of NPs is following in the order: UCNP@SiO₂-PEG-NH₂ > UCNP@SiO₂-COOH > UCNP@SiO₂-PEG. The UCL imaging and ICP-MS experimental results demonstrate that the cellular internalization of NPs is not only strongly dependent on the surface charges of NPs but also affected by the cell types.

Fig. 3 UCL images of NPs stained HeLa cells (a) and HepG-2 cells (b). The cells are stained by UCNP@SiO₂-COOH (i), UCNP@SiO₂-PEG (ii) and UCNP@SiO₂-PEG-NH₂ (iii), respectively. The concentrations of NPs are 25 μg mL⁻¹.

Fig. 4 ICP-MS measurements of cellular internalization of NPs. The cells are incubated with UCNP@SiO₂-COOH (a), UCNP@SiO₂-PEG (b) and UCNP@SiO₂-PEG-NH₂ (c), respectively. The concentrations of NPs are 25 μg mL⁻¹.

Cytotoxicity of UCNP@SiO₂ in HeLa and HepG-2 cells

The cytotoxicity of the UCNP@SiO₂ is examined by traditional MTT assay. Generally, all UCNP@SiO₂ have moderate cytotoxicity against HeLa and HepG-2 cells when the concentrations of UCNP@SiO₂ are more than 25 μg mL⁻¹ (as shown in Fig. 5). For both HeLa cells and HepG-2 cells, UCNP@SiO₂-PEG-NH₂ exhibits the highest cytotoxicity and UCNP@SiO₂-COOH has the lowest cytotoxicity. The MTT and ICP-MS experimental results indicate that the surface charge of UCNP@SiO₂ plays a critical role in interactions of UCNP@SiO₂ with their target cells. These interactions further determine intracellular uptake of UCNP@SiO₂ and their effect on the cell functions. In principle the negative membrane potential of cells likely interacts more efficiently with cationic nanoparticles due to electrostatic interactions.^17,46–48 This finding is consistent with the literature reports.^15,44 In addition, the surface charge may also affect the protein corona (a multilayered cloud of proteins held together by weak noncovalent bonds) on UCNP@SiO₂ surface, which ultimately impacts the cellular response.⁴⁹ In culture medium, the weakly charged UCNP@SiO₂-PEG may form larger and/or looser protein corona than those of highly charged UCNP@SiO₂ because the hydrodynamic diameter of UCNP@SiO₂-PEG is larger than those of UCNP@SiO₂-PEG-NH₂ and UCNP@SiO₂-COOH. The larger hydrodynamic diameter increases the steric hindrance of the nanoparticle–cell interaction, resulting in low internalization efficiency of UCNP@SiO₂-PEG and strong immune response of living cells. Therefore, the UCNP@SiO₂-PEG shows lower cellular internalization efficiency and higher cytotoxicity than those of UCNP@SiO₂-COOH.

Fig. 5 MTT assay of HeLa (a) and HepG-2 (b) cells. The cells are treated with various concentrations of UCNP@SiO₂-COOH (i), UCNP@SiO₂-PEG (ii) and UCNP@SiO₂-PEG-NH₂ (iii), respectively.

In order to evaluate the mechanism of cytotoxicity of UCNP@SiO₂, we examined the redox status of cells on exposure to different UCNP@SiO₂, intracellular releasing of metal ions and cytotoxicity of ligand. The UCNP@SiO₂ induced redox stress is tested by DCFH-DA-based assay kit.⁴¹ DCFH-DA is redox-sensitive nonfluorescent dye, which fluoresces in response to intracellular ROS. The UCNP@SiO₂-PEG-NH₂ and UCNP@SiO₂-PEG caused significant enhancement in intracellular ROS production compared to UCNP@SiO₂-PEG-COOH treated cells (as shown in Fig. 6). The physiological function of cells could be disturbed by the accumulation of intracellular ROS.^50,51 For instance, over redox stress can induce apoptosis of cells. Using Y as typical element, ICP-MS is employed to estimate the intracellular releasing of metal ions of UCNP@SiO₂ stained cells. As shown in Fig. 7, the amounts of Y in cell lysates are following in the order: UCNP@SiO₂-PEG-NH₂ stained cells > UCNP@SiO₂-PEG stained cells > UCNP@SiO₂-COOH stained cells. The released metal ions could cause damage to cell function. Furthermore, the cytotoxicity of NH₂-PEG-NH₂ is higher than that of PEG-NH₂ (as shown in Fig. S6 in the ESI†).

Fig. 6 ROS assay of HeLa and HepG-2 cells. The cells are treated with 25 μg mL⁻¹ UCNP@SiO₂-COOH (a), UCNP@SiO₂-PEG (b) and UCNP@SiO₂-PEG-NH₂ (c), respectively.

Fig. 7 ICP-MS measurements of intracellular releasing of Y ions. The cells are incubated with UCNP@SiO₂-COOH (a), UCNP@SiO₂-PEG (b) and UCNP@SiO₂-PEG-NH₂ (c), respectively. The concentrations of NPs are 25 μg mL⁻¹.

In order to obtain NPs with higher colloidal stability, excess NH₂-PEG-NH₂ molecules are normally used in the process of PEG modification to prevent interparticle cross-linking.^52,53 However, the excess NH₂-PEG-NH₂ molecules will be attracted to the surface of the NPs by electrostatic adsorption. Because of slightly acidic environments of some cell organelles (e.g., endosome, lysosome and mitochondria), the NH₂-PEG-NH₂ can be released from NP surface after UCNP@SiO₂-PEG-NH₂ uptaken by cells. Therefore, we surmise that cytotoxicity of UCNP@SiO₂ is depends on the internalization amount, enhancement in intracellular ROS production, intracellular releasing metal ions and ligand of UCNP@SiO₂.

Conclusions

In summary, UCNP@SiO₂ core@shell NPs with different surface charges and PEG ligands have been synthesized for studying the interactions of NPs with cells. Through the systematic analysis of the interactions of UCNP@SiO₂ NPs with two cell lines (HeLa and HepG-2), the experimental results demonstrate that positively charged UCNP@SiO₂-PEG-NH₂ exhibit higher cytotoxicity and cellular internalization efficiency than negatively charged UCNP@SiO₂-COOH and UCNP@SiO₂-PEG. We also posit that UCNP@SiO₂-induced redox stress and releasing of metal ions and ligands might be the underlying mechanism of charge-based cytotoxicity. Therefore, we concluded that some factors including surface charge and physicochemical stability should be considered when engineering nanomaterials with excellent biocompatibility.

Acknowledgements

The authors thank National Basic Research Program of China (no. 2011CB935800) and National Natural Science Foundation of China (Grant no. 21205113) for financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra11374h

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