Xu Wl

Biocompatibility of Fe3O4/DNR magnetic nanoparticles in the treatment of hematologic malignancies.

Purpose The objectives of this research were to assess the biocompatibility of self-assembled Fe3O4 magnetic nanoparticles (MNPs) loaded with daunorubicin (DNR), ie, (Fe3O4-MNPs/DNR), and to explore their potential application in the treatment of hematologic malignancies. Methods A hemolysis test was carried out to estimate the hematologic toxicity of Fe3O4- MNPs/DNR and a micronucleus assay was undertaken to identify its genotoxicity. Fe3O4-MNPs/ DNR were injected intraperitoneally into mice to calculate the median lethal dose (LD50). The general condition of the mice was recorded, along with testing for acute toxicity to the liver and kidneys. Results Hemolysis rates were 2.908%, 2.530%, and 2.415% after treatment with different concentrations of Fe3O4-MNPs/DNR. In the micronucleus assay, there was no significant difference in micronucleus formation rate between the experimental Fe3O4-MNPs/DNR groups and negative controls (P > 0.05), but there was a significant difference between the experimental groups and the positive controls (P < 0.05). The LD50 of the Fe3O4-MNPs/DNR was 1009.71 mg/kg and the 95% confidence interval (CI) was 769.11–1262.40 mg/kg, while that of the DNR groups was 8.51 mg/kg (95% CI: 6.48–10.37 mg/kg), suggesting that these nanoparticles have a wide safety margin. Acute toxicity testing showed no significant difference in body weight between the treatment groups at 24, 48, and 72 hours after intraperitoneal injection. The mice were all in good condition, with normal consumption of water and food, and their stools were formed and yellowish-brown. Interestingly, no toxic reactions, including instability of gait, convulsion, paralysis, and respiratory depression, were observed. Furthermore, alanine transaminase, blood urea nitrogen, and creatinine clearance in the experimental Fe3O4-MNPs/ DNR groups were 66.0 ± 28.55 U/L, 9.06 ± 1.05 mmol/L, and 18.03 ± 1.84 μmol/L, respectively, which was not significantly different compared with the control and isodose DNR groups. Conclusion Self-assembled Fe3O4-MNPs/DNR appear to be highly biocompatible and safe nanoparticles, and may be suitable for further application in the treatment of hematologic malignancies.

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

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 of 488 nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression of mdr1 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 mdr1 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.

Magnetic nanoparticle of Fe3O4 and 5-bromotetrandrin interact synergistically to induce apoptosis by daunorubicin in leukemia cells

Apoptosis is a common pathway that finally mediated the killing functions of anticancer drugs, which is an important cause of multidrug resistance (MDR). The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe3O4 (MNP(Fe3O4)) and 5-bromotetrandrin (BrTet). Analysis of the apoptosis percentage showed that combination of daunorubicin (DNR) with either MNP(Fe3O4) or BrTet exerted a potent cytotoxic effect on K562/A02 cells, while MNP(Fe3O4) and BrTet cotreatment can synergistically enhance DNR-induced apoptosis. Importantly, we confirmed that the distinct synergism effect of that composite on reverse multidrug resistance may owe to the regulation of various proliferative and antiapoptotic gene products, including P53 and caspase-3. Thus our in vitro data strongly suggests a potential clinical application of MNP(Fe3O4) and BrTet combination on CML.

Daunorubicin-loaded magnetic nanoparticles of Fe3O4 overcome multidrug resistance and induce apoptosis of K562-n/VCR cells in vivo.

Multidrug resistance (MDR) is a major obstacle to cancer chemotherapy. We evaluated the effect of daunorubicin (DNR)-loaded magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) on K562-n/VCR cells in vivo. K562-n and its MDR counterpart K562-n/VCR cell were inoculated into nude mice subcutaneously. The mice were randomly divided into four groups: group A received normal saline, group B received DNR, group C received MNPs-Fe3O4, and group D received DNR-loaded MNPs-Fe3O4. For K562-n/VCR tumor, the weight was markedly lower in group D than that in groups A, B, and C. The transcriptions of Mdr-1 and Bcl-2 gene were significantly lower in group D than those in groups A, B, and C. The expression of Bcl-2 was lower in group D than those in groups A, B, and C, but there was no difference in the expression of P-glycoprotein. The transcriptions and expressions of Bax and caspase-3 in group D were increased significantly when compared with groups A, B, and C. In conclusion, DNR-loaded MNPs-Fe3O4 can overcome MDR in vivo.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and 5-bromotetrandrine in xenograft nude-mice.

In this paper we establish the xenograft leukemia model with stable multidrug resistance in nude mice and to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe3O4 (MNP-Fe3O4) combined with daunorubicin (DNR) in vivo. Two subclones of K562 and K562/A02 cells were inoculated subcutaneously into the back of athymic nude mice (1 × 107 cells/each) respectively to establish leukemia xenograft models. Drug-resistant and sensitive tumor-bearing nude mice were assigned randomly into five groups which were treated with normal saline; DNR; NP-Fe3O4 combined with DNR; 5-BrTet combined with DNR; 5-BrTet and MNP-Fe3O4 combined with DNR, respectively. The incidence of formation, growth characteristics, weight, and volume of tumors were observed. The histopathologic examination of tumors and organs were detected. For resistant tumors, the protein levels of Bcl-2, and BAX were detected by Western blot. Bcl-2, BAX, and caspase-3 genes were also detected. For K562/A02 cells xenograft tumors, 5-BrTet and MNP-Fe3O4 combined with DNR significantly suppressed growth of tumor. A histopathologic examination of tumors clearly showed necrosis of the tumors. Application of 5-BrTet and MNP-Fe3O4 inhibited the expression of Bcl-2 protein and upregulated the expression of BAX and caspase-3 proteins in K562/A02 cells xenograft tumor. It is concluded that 5-BrTet and MNP-Fe3O4 combined with DNR had a significant tumor-suppressing effect on a MDR leukemia cells xenograft model.

Synergistic effect of the combination of nanoparticulate Fe3O4 and Au with daunomycin on K562/A02 cells

In this study, we have explored the possibility of the combination of the high reactivity of nano Fe3O4 or Au nanoparticles and daunomycin, one of the most important antitumor drugs in the treatment of acute leukemia clinically, to inhibit MDR of K562/A02 cells. Initially, to determine whether the magnetic nanoparticle Fe3O4 and Au can facilitate the anticancer drug to reverse the resistance of cancer cells, we have explored the cytotoxic effect of daunomycin (DNR) with and without the magnetic nano-Fe3O4 or nano-Au on K562 and K562/A02 cells by MTT assay. Besides, the intracellular DNR concentration and apoptosis of the K562/A02 cells was further investigated by flow cytometry and confocal fluorescence microscopic studies. The MDR1 gene expression of the K562/A02 cells was also studied by RT-PCR method. Our results indicate that 5.0 × 10−7 M nano-Fe3O4 or 2.0 × 10−8 M nano-Au is biocompatible and can apparently raise the intracellular DNR accumulation of the K562/A02 cells and increase the apoptosis of tumor cells. Moreover, our observations illustrate that although these two kinds of nanoparticles themselves could not lower the MDR1 gene expression of the K562/A02 cells, yet they could degrade the MDR1 gene level when combining with anticancer drug DNR. This raises the possibility to combine the nano-Fe3O4 or nano-Au with DNR to reverse the drug resistance of K562/A02 cells, which could offer a new strategy for the promising efficient chemotherapy of the leukemia patients.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells

In many instances, multidrug resistance (MDR) is mediated by increasing the expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic Fe3O4 nanoparticle [MNP (Fe3O4)] and MDR1 shRNA expression vector in K562/A02 cells. For stable reversal of “classical” MDR by short hairpin RNA (shRNA) aiming directly at the target sequence (3491–3509, 1539–1557, and 3103–3121 nucleotide) of MDR1 mRNA. PGC silencer-U6-neo-GFP-shRNA/MDR1 called PGY1–1, PGY1–2, and PGY1–3 were constructed and transfected into K562/A02 cells by lipofectamine 2000. After transfected and incubated with or without MNP (Fe3O4) for 48 hours, the transcription of MDR1 mRNA and the expression of P-gp were detected by quantitative real-time PCR and Western-blot assay respectively. Meanwhile intracellular concentration of DNR in K562/A02 cells was detected by flow cytometry (FCM). PGC silencer-U6-neo-GFP-shRNA/MDR1 was successfully constructed, which was confirmed by sequencing and PGY1–2 had the greatest MDR1 gene inhibitory ratio. Analysis of the reversal ratio of MDR, the concentration of daunorubicin (DNR) and the transcription of MDR1 gene and expression of P-gp in K562/A02 showed that combination of DNR with either MNP (Fe3O4) or PGY1–2 exerted a potent cytotoxic effect on K562/A02 cells, while combination of MNP (Fe3O4) and PGY1–2 could synergistically reverse multidrug resistance. Thus our in vitro data strongly suggested that a combination of MNP (Fe3O4) and shRNA expression vector might be a more sufficient and less toxic anti-MDR method on leukemia.

Effect of magnetic nanoparticles of Fe3O4 and 5-bromotetrandrine on reversal of multidrug resistance in K562/A02 leukemic cells.

This study aims to evaluate the multidrug resistance (MDR) reversal activity by magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4) and 5-bromotetrandrine (BrTet) MDR cell line K562/A02 solitarily or symphysially. The proliferation of K562 and K562/A02 cells and the cytotoxicity on peripheral blood mononuclear cells (PMBCs) were evaluated by MTT assay. Cellular accumulation of daunorubicin (DNR) was analyzed by flow cytometry. Real-time polymerase chain reaction and Western blotting analyses were performed to examine the mRNA and protein levels of mdr1, respectively. The results showed that the combination of MNPs-Fe3O4 and BrTet with effective concentrations significantly increased cytotoxicity against MDR cell line K562/A02. Both BrTet and MNPs-Fe3O4 increased the intracellular DNR accumulation in the K562/A02 cell line, and downregulated the level of mdr1 gene and expression of P-glycoprotein. Furthermore, the combination did not have significant cytotoxicity in PMBCs. We propose that MNPs-Fe3O4 conjugated with DNR and BrTet probably have synergetic effects on MDR reversal.

Synergistic effect of magnetic nanoparticles of Fe 3 O 4 with gambogic acid on apoptosis of K562 leukemia cells

Gambogic acid (GA) has a significant anticancer effect on a wide variety of solid tumors. Recently, many nanoparticles have been introduced as drug-delivery systems to enhance the efficiency of anticancer drug delivery. The aim of this study was to investigate the potential benefit of combination therapy with GA and magnetic nanoparticles of Fe3O4 (MNPs-Fe3O4). The proliferation of K562 cells and their cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscope and flow cytometry, respectively. Furthermore, real-time polymerase chain reaction and Western blotting analyses were performed to examine gene transcription and protein expression, respectively. The results showed that MNPs-Fe3O4 dramatically enhanced GA-induced cytotoxicity and apoptosis in K562 cells. The typical morphological features of apoptosis treated with GA and MNPs-Fe3O4 were observed under an optical microscope and a fluorescence microscope, respectively. The transcription of caspase-3 and bax gene in the group treated with GA and MNPs-Fe3O4 was higher than that in the GA-alone group or MNPs-Fe3O4-alone group, but the transcription of bcl-2, nuclear factor-κB, and survivin degraded as did the expression of corresponding proteins in K562 cells. Our data suggests a potential clinical application of a combination of GA and MNPs-Fe3O4 in leukemia therapy.