Reversal in multidrug resistance by magnetic nanoparticle of Fe3O4 loaded with adriamycin and tetrandrine in K562/A02 leukemic cells

Abstract

Drug resistance is a primary hindrance for efficiency of chemotherapy. To investigate whether Fe3O4-magnetic nanoparticles (Fe3O4-MNPs) loaded with adriamycin (ADM) and tetrandrine (Tet) would play a synergetic reverse role in multidrug resistant cell, we prepared the drug-loaded nanoparticles by mechanical absorption polymerization to act with K562 and one of its resistant cell line K562/A02. The survival of cells which were cultured with these conjugates for 48 h was observed by MTT assay. Using cells under the same condition described before, we took use of fluorescence microscope to measure fluorescence intensity of intracellular ADM at an excitation wavelength of488 nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression ofmdrl mRNA was measured by RT-PCR. The results showed that the growth inhibition efficacy of both the two cells increased with augmenting concentrations of Fe3O4-MNPs which were loaded with drugs. No linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe3O4-MNPs. Tet could downregulate the level of mdr-1 gene and decrease the expression of P-gp. Furthermore, Tet polymerized with Fe3O4-MNPs reinforced this downregulation, causing a 100-fold more decrease in mdrl mRNA level, but did not reduce total P-gp content. Our results suggest that Fe3O4-MNPs loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02. We propose that Fe3O4-MNPs loaded with ADM and Tet probably have synergetic effect on reversal in multidrug resistance.

Figure 1

Optical inverted microscope image of K562 incubating with different concentration of Fe₃O₄-magnetic nanoparticles polymerized with ADM for 48 h (original magnification ×600, polymeria ztion condition: 4 °C, 24 h). (a) negative control (K562). (b) K562 + ADM (50 μmol/l). (c) K562 + Fe₃O₄-MNPs0.2. (d) K562 + Fe₃O₄-MNPs-ADM0.05. (e) K562 + Fe₃O₄-MNPs-ADM0.1. (f) K562 + Fe₃O₄-MNPs-ADM0.2. (g) K562 + Fe₃O₄-MNPs-ADM0.4.

Figure 2

Optical inverted microscope image of K562/A02 incubating with different concentration of Fe₃O₄-magnetic nanoparticles polymerized with ADM for 48 h. (original magnification ×2400, polymeriaztion condition: 4 °C 24 h). (a) negative control (K562/A02). (b) K562/A02+ADM (50 μmol/l). (c) K562/A02 + Fe₃O₄-MNPs0.2. (d) K562/A02 + ADM + Tet (10 μmol/l). (e) K562/A02 + Fe₃O₄-MNPs-ADM0.05. (f) K562/A02 + Fe₃O₄- MNPs-ADM0.1. (g) K562/A02 + Fe₃O₄-MNPs-ADM0.2. (h) K562/A02 + Fe₃O₄-MNPs-ADM0.4. (i) K562/A02 + Fe₃O₄-MNPs-ADM-Tet0.05. (j) K562/A02 + Fe₃O₄-MNPs-ADM-Tet0.1. (k) K562/A02 + Fe₃O₄-MNPs-ADM-Tet0.2. (l) K562/A02 + Fe₃O₄-MNPs-ADM-Tet0.4.

Figure 3

GIE of K562 cell line by increasing concentrations Fe₃O₄-MNPs loaded with ADM or ADM and Tet. (a) After Fe₃O₄-MNPs polymerized with ADM at 37 °C for 24 h, the ADM loaded Fe₃O₄-MNPs inhibited cells proliferation more with the increasing concentrations Fe₃O₄-MNPs, so did incorporation of ADM and Tet-loaded Fe₃O₄-MNPs , R² = 0.9996 and 0.9805 respectively, P < 0.05. Comparatively, GIE of K562 acting with ADM or Fe₃O₄-MNPs single used was 73.67 ± 3.00% and 50.56 ± 6.07% respectively. (b) The similar polymerization at 4 °C for 24 h was done, R² = 0.9956 and 0.9891 respectively, P < 0.05. Comparatively, GIE of K562 acting with ADM or Fe₃O₄-MNPs single used was 70.42 ± 1.88% and 21.86 ± 3.80% respectively.

Figure 4

GIE of K562/A02 cell line by increasing concentrations Fe₃O₄-MNPs loaded with ADM or ADM and Tet. (a) After Fe₃O₄-MNPs polymerized with ADM, the ADM loaded Fe₃O₄-MNPs inhibited cells proliferation the most between Fe₃O₄-MNPs concentrations of 0.1~0.2 at 37 °C for 24 h,, so was the cell growth under the polymerization at 4 °C for 24 h. (b) There was a similar tendency in cell growth under Fe₃O₄-MNPs co-polymerized with ADM and Tet. It’s easily observed that GIE under polymerization at 4 °C was significantly higher than that at 37 °C(P < 0.05). Comparatively, GIE of K562/A02 acting with ADM, Fe₃O₄-MNPs single used or ADM and Tet was 34.69 ± 8.49%, 29.41 ± 11.21%, and 64.03 ± 10.13% respectively at 37 ° C, 51.53 ± 11.21% 6.78 ± 11.67% and 77.8 ± 12.54%, respectively at 4 °C.

Figure 5

Fluorescence view of K562/A02 intracellular ADM at a wavelength of 488 nm in scope of low power lens (×600) (Figure 7e, 7g, 7i, 7k, 7m, 7o, 7q, 7s) and high power lens (×2400) (Figure 7a~7d, 7f, 7h, 7j, 7l, 7n, 7p, 7r, 7t). The polymerization of Fe₃O₄-MNPs loaded with ADM or ADM and Tet was at 37 °C for 24 h. (a) negative control (K562/A02). (b) Fe₃O₄-MNPs 0.2. (c) ADM. (d) Tet. (e), (f) Fe₃O₄-MNPs 0.05 loaded with ADM. (i), (j) Fe₃O₄-MNPs 0.1 loaded with ADM. (m),(n): Fe₃O₄-MNPs 0.2 loaded with ADM. (q),(r) Fe₃O₄-MNPs 0.4 loaded with ADM. (g), (h) Fe₃O₄-MNPs 0.05 loaded with ADM and Tet. (k), (l) Fe₃O₄-MNPs 0.1 loaded with ADM and Tet. (o),(p): Fe₃O₄-MNPs 0.2 loaded with ADM and Tet. (s),(t) Fe₃O₄-MNPs 0.4 loaded with ADM and Tet.

Figure 7

P-glycoprotein (P-gp) expression of K562/A02 cell line by 0.1 Fe₃O₄-MNPs loaded with different concentrations of Tet (the polymerization condition: 4 °C, 48 h). (a) The mouse isotype-matched IgG2a-PE was used as negative control, P-gp was 2.61%. (b) ~(f) The monoclonal antibody (MoAb) P-gp-PE was applied in detecting P-gp. (b) The total expression P-gp of K562/A02 was 99.95%. (c) Merely 0.1 Fe₃O₄-MNPs were incubated with cells, P-gp was 99.88% with no change. (d) Merely Tet (10 μmol/l 1) were incubated with cells, P-gp was significantly decreased to 76.37%. (e) Fe₃O₄-MNPs loaded with low concentration of Tet (1 μmol/l 1), P-gp was 99.71% with no obvious change. (f) Fe₃O₄-MNPs loaded with high concentration of Tet (10 μmol/l 1), P-gp was 99.69% with no obvious change.

Figure 6

Fluorescence intensity of K562/A02 intracellular ADM. The polymerization of Fe₃O₄-MNPs loaded with ADM or ADM and Tet was at 37 °C for 24 h. (a) Fe₃O₄-magnetic nanoparticles polymerized with ADM (b) Fe₃O₄-magnetic nanoparticles polymerized with ADM and Tet. No linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe₃O₄-MNPs. The strongest fluorescence intensity was at Fe₃O₄-MNPs concentrations of 0.1~0.2. For K562/A02, ADM single or with Tet accumulated less than that loaded Fe₃O₄-MNPs.

Figure 8

MDR1 mRNA content of K562/A02 cell line by different interferes. (The polymerization condition: 4 °C, 48 h) Lane 1 was total mdr1 mRNA isolated from K562/A02 as negative control. Analyzed density of DNA was 61572. Lane 2 and Lane 3 were from the cells treated with ADM 50 μmol/l and 0.1 Fe₃O₄-MNPs respectively. The result showed 55588 and 60514 with no significant change compared with Lane 1. Lane 4 was interfered with Tet 10 μmol/l. The result showed 55588 with significant change compared with Lane 1 (P < 0.05). From lane 5 to lane 7 were MDR1 mRNA extracted from K562/A02 incubated with low Tet-, medium Tet- and high Tet-loaded 0.1 Fe₃O₄-MNPs mdr1 mRNA was significantly decreased by over 100-fold.