Influences of size of silica particles on the cellular...

JOURNAL OF NANOSCIENCE LETTERS

1

Journal of Nanoscience Letters | Volume 1 | Issue 1 | 2011 www.simplex-academic-publishers.com

© 2011 Simplex Academic Publishers. All rights reserved.

Influences of size of silica particles on the cellular endocytosis, exocytosis and cell activity of HepG2 cells

Ling Hu, Zhengwei Mao, Yuying Zhang, Changyou Gao*

MOE Key Laboratory of Macromolecular Synthesis and Functionalization,

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

*Author for correspondence: Changyou Gao, email: [email protected] Received 25 Nov 2010; Accepted 5 Jan 2011; Available Online 12 Jan 2011

Abstract

Internalization of nanoparticles into live cells closely correlates with their potential applications, functions and cytotoxicity. In this study, fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RITC) doped silica nanoparticles with four different diameters were prepared. Their average size and distribution was measured by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The optical property and zeta potential in serum containing medium of the FITC-SiO2 nanoparticles were detected, revealing that they had good stability and negative surface charge of similar value. Flow cytometry found that the uptake behavior of the FITC-SiO2 nanoparticles was dependent on their size and concentration as well as culture time. The particles mainly distributed inside the cytoplasms and endosomes, and on the cell membranes. Transportation of the silica nanoparticles into the cells was energy dependent via a clathrin-mediated endocytosis pathway, which was proved by an obvious decrease of the cellular uptake under a low temperature incubation, and NaN3, sucrose and amandatine-HCl treatments. The impact of silica nanoparticles on the HepG2 cells was assayed in terms of cytotoxicity and cell cycle as well, revealing that they had little influence on cytotoxicity even at a high particle concentration of 680 μg/ml, and no influence on the DNA synthesis at a particle dosage of 80 μg/ml for 24 h. Cellular exocytosis of the FITC-SiO2 nanoparticles was confirmed after removal of the free particles from the culture medium, which was governed by the particle size and pretreatment time too. Keywords: Silica; Nanoparticles; Cellular uptake; Endocytosis; Pathway; Cytotoxicity; Cell cycle 1. Introduction

With the rapid development of

nanotechnology, many kinds of nanomaterials have been and are being used in fields of industry and scientific researches. Especially, a wide range of engineered nanoparticles, ranging from 1-1000 nm, have been proposed to be used in nanomedicine due to their unique physical and chemical characteristics [1]. As a non-metal oxide, silica (SiO2) nanoparticles have been widely used industrially as chemical mechanical polishing, additives for drugs, cosmetics, printer toners and varnishes [2], Recently, the use of silica nanoparticles has been extended to the biomedical and biotechnological fields, such as biosensors for simultaneous assay of glucose, lactate, L-glutamate, and hypoxanthine levels in rat striatum [3], biomarkers for cellular imaging [4], cancer therapy [5], DNA and drug delivery [6-8] and enzyme immobilization [9]. Moreover, silicone dioxide is also a major component constituting the sandstorm, which is influencing many areas of the world nowadays. Therefore, the public unavoidably exposes to the

environment of silica particles, whose effect on the biological system is thereby becoming a critical issue.

Compared to other nanomaterials, the versatile silica nanoparticles stand out as an ideal platform to build up multimodal nanoparticles as well. These silica nanoparticles can be doped with desired fluorophores (visible or near infrared region) to generate fluorescent nanoparticles [10-12] which are useful for sensing, identifying and tracking intracellular structures [13-16] and local conditions such as pH [17] and redox potential [18]. Compared to those polymer-based nanoparticles, the silica nanoparticles show less aggregation, little dye leakage and are chemically inert and physically stable [19, 20]. A large number of dye molecules can be incorporated inside a single silica particle under appropriate synthetic conditions and the silica matrix provides an effective barrier keeping the trapped dye from the surrounding environment, minimizing both photobleaching and photodegradation [21]. Moreover, the silica nanoparticles can be incorporated with different bioactive molecules (such as enzymes, protein,

http://www.simplex-academic-publishers.com

mailto:[email protected]


2



peptides and chemotherapeutic drugs) following standard conjugation protocols to achieve specific biological functions [22-24]. The flexible chemistry with the silica nanoparticles provides versatile routes for surface modifications as well. For example, hydroxyl, amino, thiol and carboxyl groups can be introduced onto the particle surface with silane reagents [25-28], adding other new functionalities such as probes for magnetic resonance (MR)/radio imaging, therapeutic and targeting molecules [29]. All these features make the silica nanoparticles excellent labeling reagents for biological applications [30-32] with various targets under different conditions, from the single-molecular level to human body applications, and from in vitro diagnosis to in vivo real-time imaging [33, 34].

Although many concerns have been paid to the mesoporous silica nanoparticles in terms of cell-particle interactions [35-42], very few attempts are made to explore the interactions of non-porous silica nanoparticles with cells in terms of endocytosis, exocytosis, pathways of endocytosis and cytotoxicity etc [2, 43, 44]. Because the silica particles could be found everywhere in our life, it is important to be aware of their potential effects on human body. Therefore, basic research on the interactions of silica particles with cells should be performed, which can in turn guide their future applications in biology and medicine. In this paper, fluorescently labeled silica nanoparticles with different sizes are synthesized and their interactions with HepG2 cells are studied in terms of dynamic cellular uptake, exocytosis and cell activity. The pathways of the cellular uptake, the cytotoxicity and the potential influence on cell cycle brought by the uptake are assayed too. Since the liver is a major organ which deals with waste, here the HepG2 cells, a kind of liver source cell line, are chosen in order to disclose the interaction between the particles and liver cells. 2. Experimental 2.1. Materials

Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), amandatine-HCl, genestein, propidium iodide (PI) and RNase A (DNase-free) solution were all purchased from Sigma-Aldrich. Lyso Tracker Green DND-26 (Invigrogen, Molecular ProbesTM) was used to stain the lysosomes in cells, which gives fluorescence by excitation at 488 nm. Tetraethyl orthosilicate (TEOS) and sodium azide (NaN3) were purchased from Beijing chemical reagents company (Beijing, China). 3-Aminopropyl triethoxysilane (APS)

was purchased from Taizhou chemical plant (Hangzhou, China) and used after vacuum distillation. Human liver cancer cells (HepG2) were maintained in Dulbecco�s Modified Eagles

Medium (DMEM, Gibco) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (FBS, Sijiqing Co. Ltd., Hangzhou, China). The cells were incubated at 37 °C in a humidified atmosphere containing 5%

CO2 and used at an appropriate degree of confluence. All other reagents were analytical grade and used without further purification. Endotoxins free (<0.05 EU/ml) Millipore water with a resistivity of 18.2 M was used throughout the experiments. 2.2. Particle preparation

FITC-modified APS was prepared from a reaction between the amino group of APS and isothiocyanate of FITC under a nitrogen flow using a standard Schlenk line technique [45, 46]. In a typical synthesis process, 0.28 ml TEOS and 1 ml FITC-modified APS solution (in ethanol, the weight ratio of TEOS/APS was 0.26/0.0094) were separately injected into a mixture of 42.4 ml ethanol and 18.8 ml water under 30 oC. Condensation reaction was immediately initiated by addition of ammonia solution (0.8 ml 30 wt% of NH3.H2O) [47]. Another 2.52 ml TEOS was injected into the flask after 1 h. 5 h later, the FITC labeled silica nanoparticles (FITC-SiO2) were obtained by repeated centrifugation and washing with ethanol, and then freeze-dried. The FITC-SiO2 with different diameters were obtained by changing the weight ratio of TEOS/FTIC-APS, the volume ratios of the alcohol (or isopropanol for larger silica particles), water and ammonia solution [48]. The RITC labeled silica particles with same diameters of the FITC-SiO2 particles were similarly prepared by using RITC modified APS. 2.3. Characterization

Morphology of the FITC-SiO2 nanoparticles was characterized by a transmission electron microscope (TEM, JEOL JEM-200) with an acceleration voltage of 100 kV. Briefly, aqueous dispersion of the particles was drop-cast onto a carbon coated copper grid, and air dried. The average particle size and size distribution was also measured by dynamic light scattering (DLS, Beckman Coulter, USA) after the dried particles were re-suspended in phosphate buffered saline (PBS, pH 7.4) and DMEM/10% FBS under sonication, respectively. Zeta potential of the particles was detected by a zeta potential analyzer using an aqueous dip cell in the automatic mode (Zetasizer 3000, Malvern Instruments, Southborough, MA). The samples were prepared by mixing the particle suspension



3



in PBS with equal volume of 2 DMEM solution or 2 DMEM/20% FBS solution, resulting in a same medium as the cell incubation. The pH value of all the pre-tested solutions was adjusted to 7.4. Optical property of the FITC-SiO2 particles was detected at room temperature with a fluorescence spectrometer (LS55, Perkin Elmer precisely) after they were dispersed in PBS under sonication. The ratio of the fluorescence intensity for these particles was obtained by comparing the integrated areas of the emission peaks and was further used for normalization of the flow cytometry data. 2.4. Cell culture and uptake/exocytosis of the fluorescent silica nanoparticles

The cells were seeded onto a 24-well culture plate (Corning, New York, USA) at a density of 10×10

4 cells per well with 1 ml culture medium (DMEM containing 10% FBS, supplemented with 100 U/ml of penicillin and 100 μg/ml of streptomycin) and were cultured at

37 oC in 5% CO2 for 24 h to allow their attachment. The particles of different diameters were added into the cell culture medium at given concentrations (10 μg/ml - 640 μg/ml) and co-incubated with the cells for 24 h to explore the correlation of cellular uptake and particle concentration. For the cellular uptake dynamic analysis, at given time intervals the HepG2 cells were carefully washed 3 times with PBS, and then detached by 0.25% trypsin and re-suspended in PBS after centrifugation and washing in PBS for several times. For the exocytosis process, the particles of different diameters with a final concentration of 80 μg/ml

were first added into culture medium at 37 oC and co-incubation with the HepG2 cells for 12 h. The cells were then carefully washed 3 times with PBS to remove the free particles. This time was set as the starting point of the exocytosis process. After 1 ml fresh cell culture medium was added into each well, the cells were continually cultured until the pre-determined time intervals. The cells were carefully washed with PBS, trypsinized, re-suspended in PBS. The particles-free cultured cells were used as negative controls.

The cell suspensions were introduced into a FACSCalibur flow cytometry (BD Bioscience) with a 488-nm argon ion laser for the quantitative study of the cellular uptake/exocytosis of the FITC-SiO2 nanoparticles. The Cellquest software (BD Bioscience) was used to analyze the FACS data if not otherwise stated. The mean fluorescence intensity per cell was averaged from at least 10,000 cells. The cellular uptake/exocytosis caused by the particle internalization was assessed using the difference between the mean

fluorescence intensity of the treated cells to that of the controls. After normalized by the relative fluorescence intensity ratio of these particles respectively, the fluorescence intensity per cell was directly correlated to the uptake/exocytosis amount of the particles. The cellular uptake efficiency is defined as the ratio of the uptaking cells to the total cells, and the cellular exocytosis fraction is defined as the ratio of the removed to the uptaken particles of the cells. 2.5. Distribution of the fluorescent silica nanoparticles in HepG2 cells

To monitor the dynamic cellular uptake and distribution of the fluorescent silica nanoparticles with different diameters, all the cells were cultured on glass slides (the slides were pre-laid on the bottom of the wells in a 24 well culture plate) in the presence of 40 μg/ml

particles at 37 oC. After co-incubation for 1 h and 18 h, the free particles were aspirated and the cells were carefully washed with PBS. The cells were then stained by diluted Lyso Tracker Green solution in PBS for 10~15 min, followed by thorough washing with PBS. The images were acquired using confocal laser scanning microscopy (CLSM, LSM510, Zeiss). Section thickness was manually set smaller than 0.7 μm

and located in the middle plane of the cells. 2.6. Morphology of HepG2 cells after cellular uptake

To observe the cell morphology under scanning electron microscopy (SEM), the cells were carefully washed with PBS and fixed with 2.5% glutaraldehyde (GA) in PBS at 4 oC for 48 h after co-cultured with the FITC-SiO2 nanoparticles of different diameters for 18 h. After removal of the excessive GA, the cells were dehydrated with a graded series of ethanol, and treated with acetone and isoamyl acetate. After dried by the critical point drying method, the cells were sputter coated with an ultrathin gold layer and observed under SEM (Cambridge stereoscan 260 and FEI SIRION-100). 2.7. Cellular uptake inhibition

Various well characterized inhibiting drugs can induce certain degree of block in specific steps in the cellular uptake pathways and were chosen as the inhibitors to study the mechanism of the cellular uptake. A suitable concentration of each additive was chosen for efficacy. These inhibitive additives, including sodium azide, sucrose, amandatine-HCl and genestein, were injected into the cell culture medium prior to addition of the FITC-SiO2 nanoparticles at a final concentration of 50 mM, 200 mM, 5 mM and 0.1 mM, respectively [49, 50]. The fluorescent silica nanoparticles with



4



different diameters were then immediately added into each culture well with a final concentration of 80 μg/ml and were co-incubated at 37 oC. The cells without additives were also co-incubated with these silica nanoparticles at 4 oC. After 4 h incubation, the cells were carefully washed with PBS, trypsinized, re-suspended and introduced into a flow cytometry. The data were analyzed as mentioned above. 2.8. Toxicity of the fluorescent silica nanoparticles

The cell toxicity was measured by a methylthiazoletetrazolium (MTT) method which is a simple non-radioactive colorimetric assay [51]. Briefly, 200 μl cell suspension was seeded

onto each well of a 96-well polystyrene plate, with a final cell density of 1×10

4 /well. After 24 h, 20 μl particle suspension was added into the

culture medium with a final concentration of 190 μg/ml and 680 μg/ml, respectively. The culture medium was changed every 2 d with the fresh culture medium containing the same concentration of FITC-SiO2 nanoparticles. 20 μl

5 mg/ml MTT was added into each well and incubated for 4 h at 37 oC before measurement. After the culture medium was aspirated, the dark blue formazan crystals were dissolved with dimethyl sulfoxide. The absorbance of the supernatant was recorded at 570 nm by a microplate reader (Bio-Rad model 550). The cells initially cultured with the particles and then cultured with MTT-free medium were used as the blank references (The optical density was set as 0). The cells cultured with particles-free culture medium were set as controls. The data were analyzed with 3 parallel experiments and were expressed as mean ± standard deviation. 2.9. Cell cycle analysis [52]

HepG2 cells were seeded onto a 24-well plate at a density of 10×10

4 cells per well in 1 ml complete culture medium. The FITC-SiO2 nanoparticles of different diameters with a final concentration of 80 μg/ml were separately co-cultured with the cells at 37 oC for 24 h. The cells were carefully washed 3 times with PBS, harvested by trypsinization, washed twice with PBS and then re-suspended and fixed in 75% ice-cold ethanol under -20 °C overnight. After

washed with PBS for 1 time, the cells were re-suspended in cold PI stain buffer solution (20 μg/ml PI and 100 μg/ml DNase-free RNase A in PBS) for 30 min. The samples were analyzed by flow cytometry under a 543-nm argon ion laser. By statistically analyzing the DNA content in at least 20,000 cells, the DNA content distribution which is representative of the cell cycle distribution was obtained. Untreated HepG2 cells were used as negative controls.

2.10. Statistical analysis Data were obtained from 3 parallel

experiments and are expressed as mean ±

standard deviation (S.D.). Each experiment was repeated for 3 times to check the reproducibility if not otherwise stated. Statistical analysis was performed by two-tailed Student�s t-tests between two groups. The significant level was set as p<0.05. 3. Results 3.1. Preparation and characterization of FITC-SiO2 particles

To study the uptake of the silica nanoparticles with the equipments such as flow cytometry and CLSM et al, fluorescent labeling with good enough stability is necessary. In this work, FITC or RITC was covalently conjugated to APS, which was then reacted with TEOS to obtain the FITC-SiO2 or RITC-SiO2 nanoparticles. By variation of the fabrication conditions, the fluorescently tagged SiO2 particles with different sizes were fabricated, as typically shown in Figure 1 for the FITC-SiO2 particles. They showed a regular spherical shape and smooth surface, except for the smallest ones (Figure 1a). The mean diameter of each type of the particles was obtained by measuring about 50 particles under TEM. Table 1 summarizes the data of the four FITC-SiO2 particles with diameters of 60.3 nm (S60), 177.8 nm (S180), 368.6 nm (S370) and 592.1 nm (S600), respectively.

The sizes of the particles were also measured by DLS in PBS and DMEM/10% FBS, which are basically used to treat the particles for cell culture. Figure 2a shows that only 1 peak was recorded for each type of the particles regardless of the type of the solvents. Moreover, all the particles showed almost the same average diameters as that characterized by TEM. Slight overlapping between the S60 and S180, S370 and S600 could be observed, but the degree was not significant. In DMEM/10% FBS, the average diameters of all the particles and the polydispersity indexes were increased to some extent, implying that slight particle aggregation had occurred. It is understandable that the small particles have large surface area and tend to aggregate to reduce the surface energy, especially under the existence of proteins which decrease the surface charge intensity (Figure 2b) and may bind between the particles. Nevertheless, the aggregation was not significant and no serious overlapping of two types of particles was observed. Therefore, the colloidal dispersity and stability of the particles are good enough for cell uptake evaluation in further experiments.



5



The zeta potentials of these silica nanoparticles were measured in DMEM and DMEM/10% FBS medium, at which conditions the cells were cultured. Figure 2b shows that all the particle surfaces were negatively charged, which is resulted from the abundant -OH groups on the particle surfaces. The absolute value of the zeta potentials were decreased to some extent when the silica nanoparticles were dispersed in the DMEM/10% FBS medium, revealing the adsorption of serum proteins which are less negatively charged at physiological conditions (-5.2±1.1 mV). Therefore, the initial surfaces with

different charging degrees became almost identical due to the coverage of the proteins.

The fluorescence property of these silica particles was quantitatively measured in PBS (Figure 2c), from which the relative fluorescent intensity of the silica particles was calculated as 1.2 : 4.9 : 2.7 : 1.0 for S60, S180, S370 and S600, respectively. Since the

fluorescent dye was doped inside the particles, the fluorescent intensity of the particles was very stable and not sensitive to the environment at least for a week (over 95% of the fluorescent signals were remained, data not shown). Together with the relatively narrower distribution (Table 1), these particles can thus be used to study the influences of particle size on the cellular performance since the difference in their surface chemistry in the culture medium can be neglected. 3.2. Cellular uptake of fluorescent silica nanoparticles

Figure 3a and 3b displayed representative FACS assessments of the control and the particles-ingested HepG2 cells. Cells of the M2 group were defined as the cells uptaking nanoparticles (internalized or tightly adsorbed on the cell surfaces) because they showed fluorescent signals originated from the silica

Figure 1. TEM images of FITC-SiO2 nanoparticles with different size. (a) S60, (b) S180, (c) S370, (d) S600.

(a) (b)

(c) (d)

Table 1. Diameters of FITC-SiO2 nanoparticles measured by TEM and DLS in different medium.

Measuring method Particles size (nm)

S60 S180 S370 S600 TEM 60.3 ±3.7 177.8±6 368.6±13.8 592.1±18.4

DLS in PBS 74.5 (0.13)a 153.9 (0.16) 346.4 (0.19) 578.4 (0.21)

DLS in DMEM/10% FBS

102.3 (0.16) 196.1 (0.18) 407.1 (0.25) 662.7 (0.33)

\

a Data in the parentheses represent the polydispersity index.



6



nanoparticles. Cellular uptake (endocytosis) of the particles follows the general process identified before, in which the particles are enclosed by a portion of the cell membranes and brought into the cells in a membrane-bound vesicle [53]. In the first study of the cellular uptake of the FITC-SiO2 particles, the influence of particle concentration was assessed at a co-incubation time of 24 h (Figure 3c). The amount of internalized particles showed a positive correlation with the concentration regardless of the particle size, but their absolute amount and alteration patterns were different. Initially, the cellular uptake was increased slowly at a lower particle concentration, and then increased rapidly until ~320 g/ml. For the smaller particles such as S60 and S180, further increase of uptake was not observed, but for the S600 particles the uptake was still increased until 640 g/ml detected so far (p<0.01). Figure 3c also shows that the uptake was increased along with the particle size at a given concentration, especially above 160 g/ml.

Secondly, the dynamics of cellular uptake was monitored for a 24 h culture period by using HepG2 cells (Figure 3d) with 40 g/ml particles. It shows that the uptake monotonously increased along with the prolongation of the culture time, except for the S60 particles which were saturated at 3 h (Figure 3d, p<0.05). Moreover, decrease of the uptake amount for the

S60 particles was found in the following experimental time (3 h to 24 h, p<0.05). The cellular uptake efficiency by the HepG2 cells was found to be 80.1%, 99.6%, 97.6% and 88.6% for the S60, S180, S370 and S600, respectively, indicating that almost all the cells had ingested the S180 and S370 particles. These results reveal that all the particles can be effectively internalized or bound onto the cell surface regardless of the particle size, but the uptake amount and uptake efficiency are really size dependent. 3.3. Distribution of fluorescent silica nanoparticles in HepG2 cells

Cellular uptake and distribution of the silica nanoparticles was further studied by CLSM (Figure 4). Here the lysosomes of the cells were stained with green color, while the RITC-SiO2 particles with red color were used. Overlay of the green and red colors produced yellow, indicating the co-localization of the particles and lysosomes. At the first 1 h, some particles adhered onto the cell membranes but were hardly found inside the cytoplasms (Figure 4a-d). This result is consistent with the FCM characterization, in which the fluorescence intensity per cell was quite low at the first 1 h, since these adhered particles might be partially removed during the treatment of the cells. When the co-culture time was extended to 18 h, many

Figure 2. (a) Size distribution and (b) zeta potentials of FITC-SiO2 nanoparticles measured in PBS and DMEM/10% FBS by DLS. (c) Fluorescence spectra of FITC-SiO2 nanoparticles with a concentration of 12.2 μg/ml for S180 and 24.4 μg/ml for all other particles.

500 510 520 530 540 5500

100

200

300

400

500

600

700

800

Flu

ores

cenc

e in

tens

ity

Wavelength (nm)

S60 S180 S370 S600

Figure 2

-30

-25

-20

-15

-10

-5

0S600S370S180S60

Zet

a p

oten

tial (

mV

)

In DMEM In DMEM/10% FBS(b)

(c)

0 200 400 600 800 1000 12000

10

20

30

40

Num

ber

frac

tion

perc

enta

ge (

%)

Particle size (nm)

S60

S180S370

S600

In PBSIn DMEM/10% FBS

(a)



7



particles formed red fluorescent aggregates and accumulated both on the membranes and inside the cells. No particles were found inside the nuclei (Figure 4e-h). The existing yellow color, which was most prominent in the S370 sample, reveals that some of the particles resided in the lysosomes. Therefore, the fluorescent silica

nanoparticles with a negative surface can distribute on the cell membranes, in the cytoplasms and lysosomes. In this sense, the FCM collects the overall fluorescent signals, including that from the particles adhering on the cell membranes.

Figure 3. Representative FACS pictures of control cells (a) and cells uptaking FITC-SiO2 nanoparticles (b). M1 and M2 represent the particle-free and particles-ingested cells, respectively. Uptake of the FITC-SiO2 nanoparticles by HepG2 cells as a function of (c) concentration with a culture time of 24 h and (d) culture time with a particle concentration of 40 g/ml. Data were measured by flow cytometry and averaged to each cell. * Significant difference (p<0.05) from respective control.

0 100 200 300 400 500 600 700

0

100

200

300

400

500

600

Mea

n flu

ores

cenc

e in

tens

ity p

er c

ell

Concentation(g/ml)

S60 S180 S600

(c)

0 5 10 15 20 250

20

40

60

80

100

120

140

Mea

n flu

ores

cenc

e in

tens

ity p

er c

ell

Incubaiton time (h)

S60 S180 S370 S600

(d)

(a) (b)

Figure 3

Figure 4. CLSM images taken at (a-d) 1 h and (e-h) 18 h during the uptake process of FITC-SiO2 nanoparticles by HepG2. (a), (e) S60; (b), (f) S180; (c), (g) S370 and (d), (h) S600.

S60 S180 S370 S600

Figure 4

S60 S180 S370 S600

(a) (c)(b)

(g)(e) (h)(f)

(d)



8



3.4. Morphology of HepG2 cells after cellular uptake

After the fluorescent silica particles were incubated with HepG2 cells for 1 h, very few ones adsorbed onto the HepG2 cells and the cell morphology was not obviously changed in comparison with the particles-free cells (Figure 5a). After 18 h, a relative large number of silica particles attached onto the cell membranes in a format of clusters for the smaller ones such as S60 and S180 (Figure 5b,c). However, there were many S370 and S600 particles which singly adhered onto the cell surfaces (Figure 5d,e). 3.5. Uptake pathways of the fluorescent silica

nanoparticles There are several possible uptake

pathways for the nanoparticle internalization, including fluid-phase endocytosis, adsorptive endocytosis and receptor-mediated endocytosis. In the receptor-mediated endocytosis, certain ligands on the particle surfaces are needed to bind to the corresponding receptors on the cell surface [54]. The mechanism for cellular uptake can be mainly divided into four categories: clathrin-mediated endocytosis, caveolae-dependent endocytosis, phagocytosis and micropinocytosis [55]. Since the FITC-SiO2 nanoparticles could be efficiently internalized into the HepG2 cells, some pharmacological

Figure 5. SEM images to show cell morphology (a) before and after cellular uptake of (b) S60, (c) S180, (d) S370 and (e) S600 for 18 h, respectively.

S60 (b) S180 (c)

S370 (d) S600 (e)

(a)

Figure 6. Uptake of the FITC-SiO2 nanoparticles by HepG2 cells under different treatments. The cells were incubated with 80 g/ml FITC-SiO2 nanoparticles for 4 h under (a) 4 oC or the existence of 50 mM NaN3, and (b) with the existence of 200 mM sucrose, 5 mM amandatine-HCl and 0.1 mM genestein, respectively. # No significant difference (p > 0.05) from respective control.

(a) (b)

0

20

40

60

80

100

120

140

160

S600S370S180S60

Mea

n flu

ores

cenc

e in

tens

ity p

er c

ell control

4oC NaN

3

0

20

40

60

80

100

120

140

160

180

200

S600S370S180S60

Mea

n flu

ore

scen

c in

tens

ity p

er c

ell

Control Sucrose Amandatine-HCl Genestein

#

## #



9



inhibitors were chosen to explore the uptake mechanism in this study. First, the endocytosis is an energy-dependent process [36], indicating an inhibition of cellular uptake under low temperature or ATP-depleted (adenosine-triphosphate) environment [56]. In order to determine the silica nanoparticle internalization via endocytosis, HepG2 cells with 80 g/ml particles were co-cultured under 4 oC or in the medium having NaN3, each for 4 h. As shown in Figure 6a, a significant decrease of the fluorescence intensity (p<0.01) was observed at both conditions, leaving a value of only 10%-20% of control regardless of the particle size. For a further study of the existing pathway during cellular endocytosis, sucrose, amandatine-HCl and genestein were chosen as inhibitors for different kinds of endocytosis. It is known that sucrose and amandatine-HCl can inhibit the clathrin-mediated endocytosis by disrupting the formation of clathrin-coated vesicles [57] and preventing the budding of clathrin-coated pits [58], respectively, and genestein has an ability to block tyrosine kinases of the Src-family involved in caveolae-mediated uptake [59, 60]. Figure 6b shows that uptake of the particles by the HepG2 cells was weakened and mostly inhibited after sucrose and amandatine-HCl treatments, respectively (p<0.05). The percentage of cellular uptake was decreased to 46.8%, 56.2%, 41.3% and 41.1% after the sucrose treatment and significantly to 1.4%, 6.4%, 5.9% and 16.2% after amandatine-HCl pre-treatment for S60, S180, S370 and S600, respectively (p<0.01). By contrast, the cellular uptake was not significantly influenced (p>0.05) by the genestein treatment. These results disclose the mechanism of clathrin-mediated endocytosis for the cellular uptake of the silica nanoparticles. Since the similar results

were obtained for the four samples, this mechanism can be applied universally to the silica nanoparticles with the diameter ranging from 60 nm to 600 nm. 3.6. Toxicity of SiO2 particles

Cytotoxicity of the FITC-SiO2 particles was tested using a MTT assay. At a lower particle concentration, i.e. 190 μg/ml, all the

cells could normally proliferate and maintain >80% viability to that of the control cells during the 4 d culture (Figure 7a). In particular, the cells cultured with S60 even showed higher viability than the control after day 3 (p<0.05). When the concentration of the silica nanoparticles was improved to 680 μg/ml, relatively lower viability

was recorded for all the particles, in particular for the larger ones (Figure 7b). Especially at day 1, the cells cultured with S370 and S600 showed obviously lower viability than those cultured with S60 and S170 (p<0.05). Nevertheless, for all the samples the cell viability could still increase along with the culture time as that of the control. These results confirm that uptake of the silica particles brings rather low toxicity to the HepG2 cells. 3.7. Cell cycle analysis

Cell cycle is a series of events which can induce the cell division and duplication. In eukaryotes, the cell cycle is consist of two periods including interphase (gap1 (G1), synthesis (S) and gap2 (G2)) and mitosis (M) phase [54]. The cell cycle analysis can reflect whether the cellular uptake will influence the proliferation of live cells during their co-culture. For this study, at least 20,000 cells were collected to analyze the cell cycle status which was classified into G1, S and G2 phases. Figure 8 displays a representative FACS-histogram and

(a) (b)

Figure 7. Viability of HepG2 cells as a function of incubation time in the presence of (a) 190 μg/ml and (b) 680 μg/ml SiO2 nanoparticles. The data were normalized to that of the particle-free control cells.

1 2 3 40

20

40

60

80

100

120

Ce

ll vi

abili

ty(%

unt

reat

ed

con

tro

l cel

ls)

Incubation time (d)

S60 S180 S370 S600

1 2 3 40

20

40

60

80

100

Ce

ll vi

abi

lity

(% u

ntre

ate

d c

ont

rol c

ells

)

Incubation time (d)

S60 S180 S370 S600



10



a picture of cell cycle analysis by the Modfit software. Table 2 summarizes the relative percentage of the phases for the cells co-incubated with the FITC-SiO2 particles of different sizes. No significant difference was found for all the samples, revealing that cellular uptake of the FITC-SiO2 nanoparticles does not induce adverse effect on the cell cycle. This is consistent with the cytotoxicity assay. 3.8. Exocytosis of fluorescent silica nanoparticles

The nanoparticles may suffer from the enzymes degradation or exocytosis after cellular uptake. In the exocytosis process, the materials are packaged in secretary vesicles inside the cell and then the vesicles fuse with the plasma membrane and finally open to the exterior space [61]. For this study, the FITC-SiO2 particles were firstly co-incubated with the HepG2 cells for 12 h, and then with particles-free medium for another 12 h. Figure 9a shows that the fluorescence intensity was monotonously decreased for all the samples, suggesting that the initially internalized particles were cleaned out steadily during this culture period. To better quantify and thereby compare the ability of

particle clearance, the data in Figure 9a were transformed to exocytosis percentage and shown in Figure 9b. Clearance of the particles during the first few hours was more prominent for all the samples, with final exocytosis percentages of 63%, 67%, 58% and 38% for S60, S180, S370 and S600, respectively, revealing that smaller particles are more easily cleaned out. Moreover, clearance of the particles depends on the pre-incubation time as well. For example, if the pre-incubation time was extended to 24 h, the exocytosis percentages dropped to 50% and 10% for S60 and S600 even after a 24 h culture, respectively. 4. Discussion

The silica nanoparticles are widely used in industry, scientific research and more recently biological field along with the development of nanotechnology. People are inevitably exposed to an environment with silica nanoparticles. These tiny particles can enter the human body through skin, lung or intestinal tract and may deposit in several organs and act on living cells at the nano-level [62-64]. Therefore, it is of high significance and urgency to study the

Figure 8. A representative (a) FACS-histogram and (b) a picture of cell cycle assessment obtained from the FACS data. The data was analyzed by Modfit software.

Channels (FL2-A)0 30 60 90 120 150

Num

ber

060

012

0018

0024

00

DebrisAggregatesDip G1Dip G2Dip S

G1

G2S

(a) (b)FSC-H

0 30 60 90 120

SS

C-H

030

6090

120

R1

Table 2. Influence of cellular uptake of FITC-SiO2 nanoparticles on cell cycle of HepG2.

Sample Cell cycle distribution

G1% S% G2% control 73.1 18 8.9

S60 74.8 15.7 9.5

S180 75.3 15 9.7

S370 76.9 14.6 8.5

S600 76 15.6 8.4



11



interactions of silica nanoparticles with cells from the biological and ecological point of view.

It is known that the cellular uptake and the process of cellular delivery are influenced by various factors such as the physiochemical properties of the nanoparticles (chemical composition, size, shape and surface charge), the concentration of the nanoparticles, the incubation time, and the cell types, etc [35, 56, 65-67]. In the biological applications, the particle size is an important parameter when they are used as fluorescent probes or drug carriers [68, 69]. It has been confirmed that the particle size plays a key role in their adhesion to and interaction with biology cells. For example, the cellular uptake of both inorganic (gold [56], quantum dots [70], silica [37, 38, 71]) and organic (liposomes [72], polymer [73-76]) nanoparticles are size-dependent. However, other results showed that the extent of particle uptake is indirectly proportional to the particle size [77]. The particle size can also affect the pathway of cellular uptake [78, 79] and the cytotoxicity [39, 71, 80]. Moreover, the body circulation time and biodistribution of the nanoparticles are also influenced by their sizes [81-83]. However, there is lack of universal disciplines describing the size effect on the interactions of nanoparticles with cells, which is therefore of practical importance to be explored.

In this regard, the FITC-SiO2 nanoparticles with four different diameters were synthesized (Figure 1, Table 1). The mono dispersed silica nanoparticles could adsorb serum proteins and eventually had a similar surface charge in the culture medium (Figure 2b), so that the influence of surface chemistry can be ruled out. They showed a slight larger aggregation in DMEM/10% FBS too (Figure 2a), presumably due to the protein adsorption and weakening of

surface charge and thereby electrostatic repulsion. Since the FITC or RITC was doped inside the particles, the dye became stable and not sensitive to the environment. The fluorescent intensity of four size particles had been normalized in further quantitative comparison of the cellular uptake study (Figure 2c).

Figure 3a shows that along with the increase of particle concentration, the amount of uptaken particles was monotonously increased regardless of the particle size, which is consistent with the results reported previously [56]. It is easy to understand that if the particle concentration is too low in the bulk, the particles would have lower opportunity to meet cells and thus the cellular uptake is relatively hard to happen. Higher uptake could be achieved by increase of the particle concentration in the culture medium to some extent. Above this value, the cellular uptake does not increase again. At such as case, it is conceivable that the bottom of the culture well could be overspread with the nanoparticles and thereby the further added particles are not able to contact with the cell membranes. Since the mass of a larger particle is (rlarge/rsmall)

3 times of the smaller one, e.g. 960 times for the S600 to S60, for a similar extent of overspreading on the culture well it is quite reasonable that the larger particles are uptaken with larger amount and a higher saturation concentration. However, the number of larger particle is much smaller, e.g. the HepG2 took about 1% S600 compared with S60 at a feeding particle concentration of 640 g/ml.

The dynamic cellular uptake of the silica nanoparticles in 24 h showed the size-dependent feature too. The uptaken amount monotonously increased until 24 h, except for the S60 which decreased after 3 h. A similar decrease was also found for the PLGA

Figure 9. Time dependent exocytosis of FITC-SiO2 nanoparticles from HepG2. (a) mean fluorescence intensity per cell, and (b) fraction of exocytosed particles.

(a) (b)

0 2 4 6 8 10 120

25

50

75

100

125

Mea

n F

luor

esce

nce

Inte

nsity

per

cel

l

Exocytosis time (h)

S60 S180 S370 S600

0 2 4 6 8 10 12

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Fra

ctio

n of

nan

opar

ticle

s ex

ocyt

osed

Exocytosis time (h)

S60 S180 S370 S600



12



internalization [67]. The reason is not clear so far. One possible explanation is that the cells prefer to eat relative larger particles. Consequently, exocytosis of the particles might happen as observed in Figure 8, leading to a decrease of the particle amount inside the cells. Another possible reason is that smaller particles precipitate slower and/or have larger surface area with certain mass, leading to a lower concentration of particles which the cells can �feel�. Figure 3b,c shows also that the dynamic

cellular uptake lasted for a relatively long time, which is also reported previously [56,73]. A further study by co-incubation of HepG2 with S180 and S370 for 48 h found that the cellular uptake was increased by a factor of 13.9% and 4.9% compared with that at 24 h, respectively. This is not a big increase compared with the earlier stage before 24 h. The slope of the curves in Figure 3b,c can be regarded as the rate of cellular uptake. For the initial 3 h, the HepG2 cells took up the silica nanoparticles in a order of rate of S60 > S370 (≈ S600) > S180. The uptake rate had no obvious difference among S180, S370 and S600 from 3 h-24 h. Jin et al reported that 23 nm silica nanoparticles penetrated 81% of A549 cells, whereas the 85 nm ones penetrated only 16% over 1 h, demonstrating the cell membrane penetration of silica nanoparticles is size-dependent with a faster rate for smaller particles [71]. Chithrani et al also found that the cellular uptake of Au nanoparticles significantly increased in the first 2 h, and slowed down to reach a plateau at 4-7 h. The saturation time for cellular uptake and uptake rate of Au nanoparticles is size-dependent too, with an uptake half-life of 2.10 h, 1.90 h and 2.24 h with a rate of 622, 1294 and 417 nanoparticles per hour for the 14 nm, 50 nm and 74 nm Au nanoparticles, respectively [56].

The cellular uptake amount was size-dependent. A recent communication compared the size influence on cellular uptake of the mesoporous silica nanoparticles (MSN) of different sizes too. The amount of these particles were uptaken by HeLa cells in the order of 50 nm>30 nm>110 nm>280 nm>170 nm [37]. Chithrani et al reported that the Au nanospheres of 50 nm could be internalized most by HeLa cells among others with a diameter ranging from 14 nm - 100 nm [56]. According to Osaki�s

report, 50 nm ��glycovirus�� entered more

efficiently into cells via receptor-mediated endocytosis than smaller ones (15 nm and 5 nm) [70]. Win et al compared the cellular uptake of polystyrene (PS) nanoparticles with different diameters, showing that the PS nanoparticles internalized Caco-2 cells in an order of amount of 100 nm>200 nm>500 nm>1000 nm> 50 nm [73]. Gaumet et al found that the cellular uptake

of 100 nm poly(lactic-co-glycolic acid) (PLGA) nanoparticles into Caco-2 cells is most efficient [84]. Jin et al confirmed that the cellular uptake of carbon nanotubes with the length of 320 nm and 430 nm is more pronounced than 660 nm and 130 nm for NIH-3T3 cells [85]. These reports reveal that the size influence varies greatly and even contradicts from case to case, which might be the results of chemical and structural differences. Furthermore, the cellular uptake efficiency is slightly affected by the particle size too. The results showed that more percentage of HepG2 cells internalized S180 and S370 for a 24 h co-cultivation.

It is known that the cellular uptake can take place by the adsorptive and receptor mediated endocytosis pathways. Generally, smaller particles (0.2 μm) and larger ones (>0.5

μm) are endocytosed via pinocytosis and

phagocytosis, respectively [54]. In more detail, the clathrin-mediated endocytosis is mediated by small vesicles with a diameter of 100-150 nm, the caveolae dependent endocytosis forms small and flask-shaped pits in a diameter of 50-100 nm, and the micropinocytosis usually occurs from highly ruffled regions of the plasma membrane to form 0.5-2 µm vesicles in

diameter. In the phagocytosis the cells can bind and internalize particulate matter larger than 0.75 µm [79]. In the present study, the FITC-SiO2 nanoparticles were designed negatively charged and not decorated with any specific ligands. As a result they were all internalized in HepG2 cells by nonspecific endocytosis. However, the particle size showed no influence on the distribution and uptake mechanism of the silica nanoparticles with a diameter ranging from 60 nm to 600 nm in HepG2 cells (Figure 4-6). These nanoparticles enter into the HepG2 via the clathrin-mediated pathway, but the caveolae independent endocytosis (Figure 6b). The internalized particles mainly distributed in lysosomes and cytoplasms, but not in the cell nuclei (Figure 4). Interestingly, the amandatine-HCl reduced endocytosis percentage was weakened with the increase of particle size, which has also been observed previously by Rejman et al [86]. Lu et al reported that the fluorescent MSN with the diameters of 170 nm, 110 nm, 50 nm and 30 nm are accumulated in the perinuclear region of HeLa cells, but cannot penetrate the nucleus [37]. However, Chen et al found that 70 nm silica nanoparticles can translocate into the nucleus of epithelial cells [87]. The pathway for the cellular uptake has been well documented too. Witasp et al found that uptake of the MSN with diameters of 378 nm and 2.4 μm by macrophages is an active,

energy-dependent process through both the endocytosis and phagocytosis pathways [71].



13



Chung et al showed that the cellular uptake of 110 nm MSN by human mesenchymal stem cells and 3T3-L1 cells is via the clathrin-mediated endocytosis and actin-phagocytosis pathways [35]. Xing et al proved that the RITC-SiO2 nanoparticles can be transported into HeLa cells in part through adsorptive endocytosis and in part through fluid-phase endocytosis [88]. It is assumed that the efficient nonspecific uptake of MSN is partially due to their strong affinity to clathrin-coated vesicles [89].

The cytotoxicity of nanoparticles is of critical importance for their applications in the biological field. The nanoparticles are very reactive in the cellular environment because of their large surface area per unit mass. It has been confirmed that the cellular uptake of nanoparticles can induce a series of responses to cells to affect the cell behaviors, such as cell growth, apoptosis, adhesion, migration, differentiation, survival and tissue organization, resulting in the cytotoxicity [90, 91]. As a widely used product, the silica nanoparticles have been studied in terms of the cytotoxicity [2, 41, 65, 66, 92]. Generally, amorphous silica nano particles is considered to be safe and accredited as a food ingredient by FDA. As Brunner et al reported, the silica nanoparticles are non-toxic to human mesothelioma cells and mouse embryonic fibroblast cells under the concentrations from 0-15 mg/ml for 6 d and 30 mg/ml for 3 d [65]. However, Chang et al observed that the silica nanoparticles are slightly toxic to cells at high concentrations (138 μg/ml), especially for

normal human fibroblast cells, by retarding the cell proliferation and destroying the cell membrane [66]. Lin et al also demonstrated that the silica nanoparticles (15 nm and 46 nm) significantly reduced the viability of human alveolar epithelial cells [2]. Our previous results showed also that cellular uptake of the silica nanoparticles by HepG2 can affect cell adhesion and migration [92]. In the present study, cellular uptake of the silica nanoparticles showed low cytotoxicity to HepG2 cells with slight size-dependency and concentration-dependency. The MTT absorption of HepG2 cells appeared to be higher for S60 under a lower particle concentration (170 μg/ml). This could be explained by two possible reasons: 1) small particles may induce higher mitochondrial activity, and 2) small particles may influence cell proliferation. Since uptake of the silica nanoparticles did not affect the DNA synthesis in HepG2 cells by the cell cycle analysis (Table 2), the improved MTT absorption is mostly attributed to higher mitochondrial activity induced by small particles. One can thereby conclude that compared with other types of

nanoparticles the silica nanoparticles possess overall better safety in a cellular level.

The fate of the nanoparticles after the cellular uptake is a critical issue yet has not been well disclosed so far. One of the important events is exocytosis of the nanoparticles, which is an opposite process to the endocytosis [55, 93, 94]. The exocytosis of nanoparticles can directly influence their applications in biological system, especially for those non-degradable or decomposable particles like SiO2. In the present study, the average fluorescence intensity per cell decreased with the post culture time after the free particles were removed from the culture medium (Figure 9a), indicating that the particle expulsion from the cells takes place. There is a balance of the particle concentration inside the cells and in the culture medium which governs the endocytosis and exocytosis. After exchange with the particle-free culture medium, the particle concentration inside the cells becomes instantaneously high, and thereby drives the cellular exocytosis to such an extent that a new balance can be established. The influence of cell proliferation and apoptosis, which reduce the particle number per cell, can be ruled out since the total cell number kept unchanged during the 12 h post culture. Figure 9b reveals that the cellular exocytosis is size-dependent, e.g. larger particles like S600 are hard to be exocytosed. This phenomenon was also observed by Chithrain et al [95], in which the Au nanoparticles of smaller size could be more easily removed by HeLa cells with a faster rate and a larger dosage. Moreover, a longer pre-incubation time makes the exocytosis of the silica nanoparticles more difficult. Unlike the endocytosis, the exocytosis of nanoparticles is less studied in terms of the disciplines and mechanisms, and therefore should be paid more attention in the future study. 5. Conclusions

In summary, FITC or RITC doped SiO2 nanoparticles ranging from 60 nm to 600 nm were prepared, which showed similar surface potential (-7~-10 mV) in FBS containing culture medium. The SiO2 nanoparticles uptaken by the HepG2 cells increased along with the particle concentration and particle size. The uptaken amount for the S180, S370 and S600 was also monotonously increased along the 24 h culture time, except for the S60 which was saturated at 3 h. The uptaken amount increased along with the particle size and reached maximum for S370, and then decreased significantly. It was also found that the particles distributed inside the cytoplasms and lysosomes and on the cell membranes. Aggregates of smaller particles were



14



observed on the cell membranes too. The uptake of the silica nanoparticles was energy dependent via a clathrin-mediated endocytosis pathway regardless of the particle size. Although uptake of the particles especially the larger ones with high amount (680 μg/ml) brought some extent of

cytotoxicity, no influence was found on the cell cycle. Upon removal of the free particles from the culture medium, up to 60% of the internalized particles with a smaller size (<370 nm) were exocytosed within a 12 h post culture. These results thus disclose the basic processes and intrinsic interactions of the silica particles with cells, and may in turn guide their applications in a more safe and reasonable way. Acknowledgment

This work is financially supported by the Natural Science Foundation of China (50903069, 50873087) and Zhejiang Provincial Natural Science Foundation of China (Z4090177).

References 1. O.V. Salata, J. Nanobiotechnol. 2 (2004) 3. 2. W.S. Lin, Y. Huang, X.D. Zhou, Y.F. Ma,

Toxicol. Appl. Pharmacol. 217 (2006) 252. 3. F.F. Zhang, Q. Wan, C.X. Li, X.L. Wang,

Z.Q. Zhu, Y.Z. Xian, L.T. Jin, K. Yamamoto, Anal. Bioanal. Chem. 380 (2004) 637.

4. S. Santra, P. Zhang, K. Wang, R. Tapec, W. Tan, Anal. Chem. 73 (2001) 4988.

5. L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R. Sershen, B. Rivera, R.E. Price, J.D. Hazle, N.J. Halas, J.L. West, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 13549.

6. D.J. Bharali, I. Klejbor, E.K. Stachowiak, P. Dutta, I. Roy, N. Kaur, E.J. Bergey, P.N. Prasad, M.K. Stachowiak, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 11539.

7. R.A. Gemeinhart, D. Luo, W.M. Saltzman, Biotechnol. Prog. 21 (2005) 532.

8. N. Venkatesan, J. Yoshimitsu, Y. Ito, N. Shibata, K. Takada, Biomaterials 26 (2005) 7154.

9. M. Qhobosheane, S. Santra, P. Zhang, W. Tan., Analyst 126 (2001) 1274.

10. H. Ow, D.R. Larson, M. Srivastava, B.A. Baird, W.W. Webb, U. Wiesner, Nano Lett. 5 (2005) 113.

11. X. Zhao, R. Tapec-Dytioco, W. Tan, J. Am. Chem. Soc. 125 (2003) 11474.

12. X. Zhao, L.R. Hilliard, S.J. Menchery, Y. Wang, R.P. Bagwe, S. Jin, W. Tan, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 15027.

13. Hoshino, K. Fujioka, T. Oku, S. Nakamura, M. Suga, Y. Yamaguchi, K. Suzuki, M.

Yasuhara, K. Yamamoto, Microbiol. Immunol. 48 (2004) 985.

14. J. Jaiswal, E. Goldman, H. Mattoussi, S. Simon, Nature Meth. 1 (2004) 73.

15. X. Michalet, F. Pinaud, L. Bentolila, J. Tsay, S. Doose, J. Li, G. Sundaresan, A.M.Wu, S.S. Gambhir, S. Weiss, Science 307 (2005) 538.

16. F. Chen, D. Gerion, Nano Lett. 4 (2004) 1827.

17. Burns, P. Sengupta, T. Zedayko, B. Baird, U. Wiesner, Small 2 (2006) 723

18. S. Clarke, C. Hollmann, Z. Zhang, D. Suffern, S. Bradforth, N. Dimitrijevic, W.G. Minarik, J.L., Nature Mater. 5 (2006) 409.

19. W. Tan, K. Wang, X. He, X.J. Zhao, T. Drake, L. Wang, R.P. Bagwe, Med. Res. Rev. 24 (2004) 621.

20. H.K. Kim, S.J. Kang, S.K. Choi, Y.H. Min, C.S. Yoon, Chem. Mater. 11 (1999) 779.

21. T.K. Jain, I. Roy, T.K. De, A.N. Maitra, J. Am. Chem. Soc. 120 (1998) 11092.

22. J. Cordek, X. Wang, W. Tan, Anal. Chem. 71 (1999) 1529.

23. X.H. Fang, X. Liu, S. Schuster, W. Tan, J. Am. Chem. Soc. 121 (1999) 2921.

24. S. Huh, J.W. Wiench, J.-C. Yoo, M. Pruski, V.S.-Y. Lin, Chem. Mater. 15 (2003) 4247.

25. Walcarius, M. Etienne, B. Lebeau, Chem. Mater. 15 (2003) 2161.

26. F. Cagnol, D. Grosso, C. Sanchez, Chem. Commun. (2004) 1742.

27. L. Han, Y. Sakamoto, O. Terasaki, Y. Li, S. Che, J. Mater. Chem. 17 (2007) 1216.

28. P. Sharma, S. Brown, G. Walter, S. Santra, B. Moudgil, Adv. Colloid Interface Sci. 123 (2006) 471.

29. J.E. Smith, L. Wang, W. Tan, Trends Anal. Chem. 25 (2006) 848.

30. R.P. Bagwe, L.R. Hilliard, W. Tan, Langmuir 22 (2006) 4357.

31. G. Yao, L. Wang, Y. Wu, J. Smith, J. Xu, W. Zhao, E. Lee, W. Tan, Anal. Bioanal. Chem. 385 (2006) 518.

32. X. Zhao, R.P. Bagwe, W. Tan, Adv. Mater. 16 (2004) 173.

33. N.C. Tansil, Z. Gao, Nano Today 1 (2006) 28.

34. S.G. Penn, L. He, M.J. Natan, Curr. Opin. Chem. Biol. 7 (2003) 609.

35. T.H. Chung, S.H. Wu, M. Yao, C.W. Lu, Y.S. Lin, Y. Hung, C.Y. Mou, Y.C. Chen, D.M. Huang, Biomaterials 28 (2007) 2959.

36. J. Lu, M. Liong, S. Sherman, T. Xia, M. Kovochich, A.E. Nel, J.I. Zink, F. Tamanoi, Nanobiotechnol. 3 (2007) 89.

37. F. Lu, S.H. Wu, Y. Hung, C.Y. Mou, Small 5 (2009) 1408.

38. E. Witasp, N. Kupferschmidt, L. Bengtsson, K. Hultenby, C. Smedman, S. Paulie, A.E.



15



Garcia-Bennett, B. Fadeel, Toxicol. Appl. Pharmacol. 239 (2009) 306.

39. H. Vallhov, S. Gabrielsson, M. Stromme, A. Scheynius, A.E. Garcia-Bennett, Nano Lett. 7 (2007) 3576.

40. I.I. Slowing, J.L. Vivero-Escoto, C.W. Wu, V.S.Y. Lin, Adv. Drug Deliv. Rev. 60 (2008) 1278.

41. Z. Tao, M.P. Morrow, T. Asefa, K.K. Sharma, C. Duncan, A. Anan, H.S. Penefsky, J. Goodisman, A.K. Souid, Nano Lett. 8 (2008) 1517.

42. S.P. Hudson, R.F. Padera, R. Langer, D.S. Kohane, Biomaterials 29 (2008) 4045.

43. S. Santra, B. Liesenfeld, D. Dutta, D. Chatel, C.D. Batich, W. Tan, B.M. Moudgil, R.A. Mericle, J. Nanosci. Nanotechnol. 5 (2005) 899.

44. S. Santra, H. Yang, D. Dutta, J.T. Stanley, P.H. Holloway, W. Tan, B.M. Moudgil, R.A. Mericle, Chem. Commun. (Cambridge) 25 (2005) 3144.

45. N.A.M. Verhaegh, A.V. Blaaderen, Langmuir 10 (1994) 1427.

46. A.V. Blaaderen, V. Vrij, Langmuir 8 (1992) 2921.

47. K. Han, Z. Xiang, M. Li, H. Zhang, J. Zhang, B. Yang, Colloid. Surf. A 280 (2006) 169.

48. W. Wang, B. Gu, L. Liang, W. Hamilton, J. Phys. Chem. B 107 (2003) 3400.

49. Z.P. Xu, M. Niebert, K. Porazik, T.L. Walker, H.M. Cooper, A.P.J. Middelberg, P.P. Gray, P.F. Bartlett, G.Q. Lu, J. Control. Release 130 (2008) 86.

50. M.F. Yanze, W.S. Lee, K. Poon, Piquette-Miller, M. and Macgregor-Jr, R. B. Biochem. 42 (2003) 11427.

51. T. Mossmann, J. Immunol. Methods 48 (1983) 631.

52. J.S. Park, T.H. Han, K.Y. Lee, S.S. Han, J.J. Hwang, D.H. Moon, S.Y. Kim, Y.W. Cho, J. Control. Release 115 (2006) 37.

53. J.W. Kimball, Biology, 6th ed., Dubuque, IA, Wm. C. Brown Publishers (1994).

54. X.M. Tang, Medical cell biology, Science. Press of China, 1st ed., Beijing (2003).

55. G.J. Doherty, H.T. McMahon, Annu. Rev. Biochem. 78 (2009) 857.

56. B.D. Chithrani, A.A. Ghazani, C.W.C. Warren, Nano Lett. 6 (2006) 662.

57. J.J.H. Chu, M.L. Ng. J. Virol. 78 (2004) 10543.

58. D.G. Perry, G.L. Daugherty, W.J.I. Martin, J. Immun. 162 (1996) 380.

59. N.S. Dangoria, W.C. Breau, H.A. Anderson, D.M. Cishek, L.C. Norkin, J. Gen. Virol. 77 (1996) 2173.

60. R.G. Parton, B. Joggerst, K. Simons, J. Cell Biol. 127 (1994) 1199.

61. H. Lodish, A. Berk, L. Zipursky, P. Matsudaira, D. Baltimore, J. Darnell, Molecular cell biology, 4th ed., W. H. Freeman, New York (2000).

62. G. Oberdörster, A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J.C. Carter, B. Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, H. Yang, Part Fibre Toxicol. 2 (2005) 8.

63. G. Oberdörster, E. Oberdörster, J.

Oberdörster, Environ. Health Perspect. 113 (2005) 823

64. C. Medina, M.J. Santos-Martinez, A. Radomski, O.I. Corrigan, M.W. Radomski, J. Pharmacol. 150 (2007) 552.

65. T.J. Brunner, P. Wick, P. Manser, P. Spohn, R.N. Grass, L.K. Limbach, A. Bruinink, W.J. Stark, Environ. Sci. Technol. 40 (2006) 4374.

66. J.S. Chang, K. Liang, B. Chang, D.F. Hwang, Z.L. Kong, Environ. Sci. Technol. 41 (2007) 2064.

67. M.S. Cartiera, K.M. Johnson, V. Rajendran, M.J. Caplan, W.M. Saltzman, Biomaterials 30 (2009) 2790.

68. P. Tallury, K. Payton, S. Santra, Nanomedicine 3 (2008) 579.

69. A.M. Smith, H.W. Duan, A.M. Mohs, S.M. Nie, Adv. Drug. Delivery Rev. 60 (2008) 1226.

70. F. Osaki, T. Kanamori, S. Sando, T. Sera, Y. Aoyama, J. Am. Chem. Soc. 126 (2004) 6520.

71. Y.H. Jin, S. Lohstreter, D.T. Pierce, J. Parisien, M. Wu, C. Hall, J.X. Zhao, Chem. Mater. 20 (2008) 4411.

72. S. Chono, T. Tanino, T. Seki, K. Morimoto, J. Pharm. Pharmacol. 59 (2007) 75.

73. K.Y. Win, S.S. Feng, Biomaterials 26 (2005) 2713.

74. S.W. Pang, H.Y. Park, Y.S. Jang, W.S. Kim, J.H. Kim, Colloids Surf. B 26 (2002) 213.

75. C. Foged, B. Brodin, S. Frokjaer, A. Sundblad, Int. J. Pharm. 298 (2005) 15315.

76. C. Cortez, E. Tomaskovic-Crook, A.P.R. Johnston, A.M. Scott, E.C. Nice, J.K. Heath, F. Caruso, ACS Nano 1 (2007) 93.

77. M.P. Desai, V. Labhasetwar, E. Walter, R.J. Levy, G.L. Amidon, Pharm. Res. 14 (1997) 1568.

78. Z.J. Wang, C. Tiruppathi, R.D. Minshall, A.B. Malik, ACS Nano 3 (2009) 4110.

79. L. Chen, H. Li. R. Zhao, J.W. Zhu, J. Clinical Oncol. 8 (2009) 360.

80. D. Napierska, L.C. Thomassen, V. Rabolli, D. Lison, L. Gonzalez, M. Kirsch-Volders, J.A. Martens, P.H. Hoet, Small 5 (2009) 846.

81. E. Chang, N. Thekkek, W.W. Yu, V.L. Colvin, R. Drezek, Small 2 (2006) 1412.



16



82. M. Geiser, M. Casaulta, B. Kupferschmid, H. Schulz, M. Semmler-Behnke, W. Kreyling, Am. J. Respir. Cell Mol. Biol. 38 (2008) 371.

83. E. Sadauskas, H. Wallin, M. Stoltenberg, U. Vogel, P. Doering, A. Larsen, G. Danscher, Part. Fibre Toxicol. 4 (2007) 10.

84. M. Gaumet, R. Gurny, F. Delie, Eur. J. Pharm. Sci. 36 (2009) 465.

85. H. Jin, D.A. Heller, R. Sharma, M.S. ACS Nano 3 (2009) 149.

86. J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Biochem. J. 377 (2004) 159.

87. M. Chen, A. von Mikecz, Exp. Cell Res. 305 (2005) 51.

88. X. Xing, X. He, J. Peng, K. Wang, W. Tan, J. Nanosci. Nanotechnol. 5 (2005) 1688.

89. D.M. Huang, Y. Hung, B.S. Ko, S.C. Hsu, W.H. Chen, C.L. Chien, C.P. Tsai, C.T. Kuo, J.C. Kang, C.S. Yang, C.Y. Mou, Y.C. Chen,. FASEB J. 19 (2005) 2014.

90. D.R. Absolom, W. Zingg, A.W. J. Biomed. Mater. Res. 21 (1987) 161.

91. T.A. Haas, E.F. Plow, Curr. Opin. Cell Biol. 6 (1994) 656.

92. Y. Zhang, L. Hu, C. Gao, Acta Polymerica Sinica. 8 (2009) 815.

93. X.H.N.Xu, W.J. Brownlow, S.V. Kyriacou, Q. Wan, J.J. Biochem. 43 (2004) 10400.

94. H. Fischer, L. Li, K.S. Pang, W.C.W. Chan, Adv. Funct. Mater. 16 (2006) 1299.

95. B.D. Chithrani, W.C.W. Chan, Nano Lett. 7 (2007) 1542.


Influences of size of silica particles on the cellular...

Documents

Transcript of Influences of size of silica particles on the cellular...