Lung adenocarcinoma cells on glutathione endocytosis of nano-Fe3O4

Key words human lung adenocarcinoma cells; glutathione; Fe3O4; endocytosis

Abstract: Objective To observe the in vitro human lung adenocarcinoma cells (SPC-A1) on glutathione (GSSG) Fe3O4 nanoparticles modified with endocytosis and with incubation temperature, time and concentration. Methods Transmission electron microscopy (TEM) and Prussian blue staining cells SPC-A1 Fe3O4 nanoparticles modified GSSG endocytosis. By atomic absorption spectrometry (AAS) method is the amount of SPC-A1 cell adhesion and endocytosis amount of quantitative analysis. Results TEM results showed that cultured 24h at 4 ℃ after the GSSG-modified Fe3O4 nanoparticles adsorbed on the SPC-A1 cells; 37 ℃ 1h cultured cells SPC-A1 after the endocytosis of modified nano-Fe3O4 GSSG significantly increased the number of cells. AAS analysis showed that, SPC-A1 cell surface adhesion amount of Fe3O4 nanoparticles with the incubation time and concentration were positively correlated. GSSG Fe3O4 nanoparticles modified with low concentrations (01 or 04mg/ml), the endocytic capacity in saturated after 24h; GSSG Fe3O4 nanoparticles modified with higher concentration (07 or 10mg/ml), the volume of endocytosis reached after 72h saturated, the largest single cell up to the endocytic 160pg. Conclusion SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis requires energy, endocytosis is dependent on concentration and incubation time.

Key words: Human lung adenocarcinoma cells; glutathione; Fe3O4; endocytosis

Endocytosis of lung adenocarcinoma cell on glutathione modified magnetite (Fe3O4) nanoparticles
YAN Zhubing, MA Yongjie, GU Hongchen, et al.

Institute of Micro and Nano Science and Technology, Shanghai Jiaotong University (Shanghai 200030, China)

Abstract: Objective To study the relationship between endocytosis of human lung adenocarcinoma cell line SPC-A1 modified by oxidized Glutathione (GSSG) and culture conditions including culture temperature, time and concentration.Methods The uptake of GSSG-modified Fe3O4 nanoparticles by SPC-A1 cell was observed by transmission electron microscopy (TEM) and Prussian blue staining.The extracellular adhesion or intracellular uptake of GSSG-modified Fe3O4 nanoparticles were measured by atom absorb spectrum (AAS). Results TEM results indicated that GSSG-modified Fe3O4 nanoparticles adhered on the membrane of SPC-A1 cell after bEing cultured at 4 ℃ for 1h or 24h, but SPC-Al cell could took up nanoparticles particles after cultured at 37 ℃ for 1h.Prussian blue staining indicated that SPC-Al cells containing GSSG-modified Fe3O4 nanoparticles were much more with the prolongation of culture time or the incre (Chinese papers Union www.lwlm.com order) ase of concentration.AAS analysis indicated that the extracellular adhesion of GSSG-modified magnetic particles was depended on culture time and concentration.The uptake amount of GSSG-modiied Fe3O4 nanoparticles by SPC-A1 cell was saturated after 24h when the nanoparticles in low concentration (0.1mg/ml or 0.4mg/ml), the uptake was saturated after 72h when the nanoparticles in high concentration (0.7mg/ml or 1.0mg/ml). The maximal amount per SPC-A1 cell could reach 160pg.Conclusion The uptake of GSSG-modified Fe3O4 nanoparticles by SPC-A1 cell is energy needed.It depends on the concentration of nanoparticles and saturates at certain time.

Key words: human lung adenocarcinoma cell; glutathione; magenetite (Fe3O4); endocytosis

Oxidized glutathione (GSSG) by the reduced glutathione (GSH) from oxidation, which is widely distributed in 人体各器官 inside. In vivo, GSSG reductase and GSH in the role of the state can be transformed into each other 〔1,2〕 balance, which Fe3O4 nanoparticles modified by GSSG can improve its biocompatibility. Cell endocytosis Fe3O4 nanoparticles can affect the amount of intracellular hyperthermia 〔3〕, magnetic resonance imaging results 〔4〕. In this paper, transmission electron microscopy, Prussian blue staining, atomic absorption spectrometry method for qualitative or quantitative observation of human lung adenocarcinoma SPC-A1 cells to GSSG Fe3O4 nanoparticles modified with endocytosis and with incubation temperature, time, concentration of each other.

1 Materials and methods

11 Synthesis of Fe3O4 magnetic nanoparticles GSSG surface modified nanoparticles: weighing 705g FeCl3 · 6H2O and 297g FeCl2 · H2O, stirred on the magnetic stirrer. Temperature rises to 80 ℃, add 20ml concentrated ammonia, mechanical stirrer reaction 30min, adding 02gGSSG (Shanghai Chemical Reagent Company), under the conditions of reaction at 85 ℃ 1h, remove ammonia, the resultant separated with a permanent magnet repeatedly washed with deionized water 5 times, a steady GSSG modified nano Fe3O4, the concentration of 52mg/ml.

12 GSSG characterization of Fe3O4 nanoparticles modified transmission electron microscope (Philips-TECNAI20S-TWIN)-generated GSSG observed Fe3O4 nanoparticles modified morphology, X-ray diffraction (Bruker-AXS X-ray rotating anode powder diffractometer) measurements its crystal structure.

TEM 13 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis of human lung adenocarcinoma cells, Institute of Cell Biology, Chinese Academy of Sciences) were cultured in the recovery of 10% fetal calf serum containing RPMI-1640 medium (American Haike Long Company), placed CO2 incubator (Thermo Corporation) and maintained in culture 37 ℃, 5% CO2. After 24h culture medium replaced with 02mg/ml GSSG Fe3O4 nanoparticles modified culture medium, the growth status of the same dish 3 SPC-A1 cells were cultured according to the following conditions continue: 4 ℃ cultured 1h, 37 ℃ incubation 1h, 4 ℃ incubated 24h. Were collected three kinds of cell culture conditions, using the literature method of sample preparation 〔5〕 observed.

14 GSSG Fe3O4 nanoparticles modified in SPC-A1 cell surface adhesion determination of the amount of SPC-A1 cells to 5 × 105 per well the number of seeded in 6-well culture plates, culture medium volume was 2ml. Cultured at 37 ℃ for 24h, remove the culture medium, the replacement for the 02mg / m.
GSSG Fe3O4 nanoparticles modified culture medium, were cultured at 4 ℃ after the determination of GSSG 0,3,6,12,24 h Fe3O4 nanoparticles modified SPC-A1 cells in the amount of surface adhesion. Also set the concentration of nano-Fe3O4 01 and 04mg/ml, measured the amount of adhesion after 24h culture. Determination by reference 〔6〕.

15 Prussian blue staining at 37 ℃, in the exponential phase of the SPC-A1 cells and 02mg/mlGSSG Fe3O4 nanoparticles modified the culture medium followed by 12 ~ 24h, using Prussian blue staining cells Fe3 + 〔7〕. In addition to set 01 or nano-Fe3O4 concentration of 10mg/ml, Prussian culture stained after 24h. Chinese papers Alliance WWW.LWLM.COM finishing.

16 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic contents at 37 ℃, SPC-A1 cells to 5 × 105 per well the number of seeded in 6-well culture plates, culture medium volume was 2ml. After 24h culture, the culture medium replaced by Fe3O4 nanoparticles modified with GSSG in the culture medium. Concentration of nano-Fe3O4 were 05,1,15,20 mg / ml. Four kinds of concentration were cultured 0,3,6,12,24,48,72 h, determination of SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity. Determination by reference 〔6〕.

2 Results

21 GSSG characterization of Fe3O4 nanoparticles modified (Figure 1) transmission electron microscope, GSSG Fe3O4 nanoparticles modified for the uniform spherical particles with narrow size distribution, particle size is 7 ~ 15nm. GSSG-modified nano-Fe3O4 magnetic fluid formed in aqueous solution, ultrasonic treatment has a good dispersion and stability. XRD spectra of the samples that the samples are cubic spinel structure.

Figure 1 GSSG Fe3O4 nanoparticles modified TEM image (scale to 20nm) (omitted)

22 transmission electron microscope at 4 ℃ SPC-Al cells and 02mg/mlGSSG Fe3O4 nanoparticles modified the culture medium after 1h, nano Fe3O4 SPC-A1 cell adhesion to the surface is not internalized into the SPC-Al cells (Fig. 2a), After 1h at 37 ℃ culture GSSG Fe3O4 nanoparticles modified to internalize into SPC-A1 cells (Fig. 2b), incubated at 4 ℃ for 24h, nano Fe3O4 still not internalized into SPC-A1 cells (Fig. 2c).

Note: "" indicates that GSSG-modified magnetic nanoparticles; a: 4 ℃ after 1h culture TEM image; b: 37 ℃ after 1h incubation TEM image; c: 4 ℃ after 24h incubation TEM images

Under different culture conditions Figure 2 SPC-A1 cells by transmission electron microscopy images (omitted)

23 GSSG Fe3O4 nanoparticles modified in the SPC-Al cell surface adhesion amount of GSSG Fe3O4 nanoparticles modified in SPC-A1 cell surface adhesion increased with the prolongation of culture time increased, GSSG Fe3O4 nanoparticles modified cells in SPC-A1 Adhesion is dependent on GSSG concentration of Fe3O4 nanoparticles modified, both showed a linear relationship.

24 Prussian blue staining when the culture medium GSSG Fe3O4 nanoparticles modified when the concentration of 02mg/ml, endocytosis after 24h culture GSSG Fe3O4 nanoparticles modified the SPC-A1 was more than the number of cultured cells after 12h (Figure 3a). When the incubation time was 24h, with medium to increase the concentration of nano-Fe3O4, endocytic GSSG Fe3O4 nanoparticles modified by the number of SPC-A1 cells was significantly increased (Figure 3b, Figure 3c, Figure 3d)

25 SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity (Chinese papers Union www.lwlm.com order) SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytosis is dependent on GSSG concentration of Fe3O4 nanoparticles modified, Fe3O4 nanoparticles modified with the GSSG concentration corresponding endocytosis also increased. Fe3O4 nanoparticles modified when the GSSG concentration of 01 and 04mg/ml, the cultured cells SPC-A1 after 24h endocytosis reached saturation; Fe3O4 nanoparticles modified when the GSSG concentration of 07 and 10mg/ml, the SPC-A1 after 72h culture endocytic cells reached saturation. SPC-A1 cells to a single GSSG Fe3O4 nanoparticles modified up to the endocytic capacity 160pg.

Note: a: GSSG concentration of Fe3O4 nanoparticles modified 02mg/ml, incubation time 12h;

b: GSSG concentration of Fe3O4 nanoparticles modified 02mg/ml, incubation time 24h;

c: GSSG concentration of Fe3O4 nanoparticles modified 01mg/ml, incubation time 24h;

d: GSSG concentration of Fe3O4 nanoparticles modified 10mg/ml, incubation time 24h

Figure 3 Prussian blue staining images (200 ×) (omitted)

3 Discussion

In this paper, transmission electron microscopy at 4 ℃ for 1 or 24h after training, GSSG Fe3O4 nanoparticles modified only SPC-A1 cell adhesion to the surface, not internalized into the cells. And 37 ℃ for 1h after training, SPC-A1 cells are able to internalize the GSSG-modified nano-Fe3O4. SPC-A1 cells confirmed that GSSG Fe3O4 nanoparticles modified without the energy of adhesion, the role of internalization requires energy. This paper studies the impact of GSSG at 4 ℃ Fe3O4 nanoparticles modified SPC-A1 cell adhesion to the surface of the relevant factors. The results showed that the adhesion amount of time and with the culture medium Fe3O4 nanoparticles modified with the concentration of GSSG showed a linear relationship. Many foreign scholars have studied different cell Fe3O4 nanoparticles of different endocytosis. The results show that cells of Fe3O4 nanoparticle endocytosis and cell types, Fe3O4 nanoparticles surface modification, size _ the culture time, Fe3O4 nanoparticles concentration, temperature and other factors 〔8,9〕. This Prussian blue staining results showed that with prolonged incubation time or increasing the concentration of Fe3O4 nanoparticles in SPC-A1 significantly increased the number of cells, internalization of the GSSG-modified nano-Fe3O4 are all located in the cytoplasm. AAS results show that Fe3O4 nanoparticles modified with the concentration of GSSG increased iron content in individual cells increased, the reason may be to increase the concentration of nano-particles, nano-particles increased the adhesion of nanoparticles led to an increase in internalization. SPC-A1 cells to GSSG Fe3O4 nanoparticles modified endocytic capacity will be saturated with culture time and cell growth may be a single cellular iron status and on the capacity of saturation.

References


〔1〕 Jordan A, Scholz R, Klaus MH, et al.Presentation of a new magnetic field therapy system for the treatment of human solid tumors with magnetic fluid hyperthermia [J]. Journal of Magnetism and Magnetic Materials, 2001,225:118