Biocompatibility of magnetic Fe3O4 nanoparticles and their cytotoxic effect on MCF-7 cells.

BACKGROUND: The objective of this study was to evaluate the synthesis and biocompatibility of Fe3O4 nanoparticles and investigate their therapeutic effects when combined with magnetic fluid hyperthermia on cultured MCF-7 cancer cells. METHODS: Magnetic Fe3O4 nanoparticles were prepared using a coprecipitation method. The appearance, structure, phase composition, functional groups, surface charge, magnetic susceptibility, and release in vitro were characterized by transmission electron microscopy, x-ray diffraction, scanning electron microscopy-energy dispersive x-ray spectroscopy, and a vibrating sample magnetometer. Blood toxicity, in vitro toxicity, and genotoxicity were investigated. Therapeutic effects were evaluated by MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] and flow cytometry assays. RESULTS: Transmission electron microscopy revealed that the shapes of the Fe3O4 nanoparticles were approximately spherical, with diameters of about 26.1 ± 5.2 nm. Only the spinel phase was indicated in a comparison of the x-ray diffraction data with Joint Corporation of Powder Diffraction Standards (JCPDS) X-ray powder diffraction files. The O-to-Fe ratio of the Fe3O4was determined by scanning electron microscopy-energy dispersive x-ray spectroscopy elemental analysis, and approximated pure Fe3O4. The vibrating sample magnetometer hysteresis loop suggested that the Fe3O4nanoparticles were superparamagnetic at room temperature. MTT experiments showed that the toxicity of the material in mouse fibroblast (L-929) cell lines was between Grade 0 to Grade 1, and that the material lacked hemolysis activity. The acute toxicity (LD(50)) was 8.39 g/kg. Micronucleus testing showed no genotoxic effects. Pathomorphology and blood biochemistry testing demonstrated that the Fe3O4 nanoparticles had no effect on the main organs and blood biochemistry in a rabbit model. MTT and flow cytometry assays revealed that Fe3O4 nano magnetofluid thermotherapy inhibited MCF-7 cell proliferation, and its inhibitory effect was dose-dependent according to the Fe3O4 nano magnetofluid concentration. CONCLUSION: The Fe3O4 nanoparticles prepared in this study have good biocompatibility and are suitable for further application in tumor hyperthermia.

3,3'-Diiodothyronine sulfate cross-reactive material (compound W) in human newborns.

BACKGROUND: Thyrosulfoconjugation appears to facilitate fetal-to-maternal transfer of 3,3'-diiodothyronine-sulfate (T(2)S). Elevated maternal levels of T(2)S cross-reactive material (compound W) are found in humans, with higher levels found in venous cord blood than in arterial samples. These findings are consistent with the postulate that the placenta plays an essential role in compound W production. METHODS: Serum compound W levels were measured by a T(2)S-specific radioimmunoassay in 60 serum samples from newborns with hyperbilirubinemia, age 1-30 d. In addition, 59 maternal serum samples, from day 1 to day 7 after uneventful deliveries, were studied. RESULTS: As compared with day 1, at day 5, the mean (±SE) compound W level fell to 43.5 ± 6.8% (decay half-life (t(1/2)) = 4.12 d) and to 33.7 ± 4.6% (decay t(1/2) = 2.82 d) in the newborn and maternal groups, respectively. In the mothers, the level continued to decline along the same slope through day 7. In the newborns, however, the mean compound W level entered a slower phase of decay after the fifth day with a decay t(1/2) = 10.9 d. CONCLUSION: Compound W is cleared at similar rates in newborn and postpartum maternal sera. This is consistent with the postulate that compound W is produced in the placenta.

Preparation and biodistribution of 188Re-labeled folate conjugated human serum albumin magnetic cisplatin nanoparticles (188Re-folate-CDDP/HSA MNPs) in vivo.

BACKGROUND: The purpose of this study was to develop intraperitoneal hyperthermic therapy based on magnetic fluid hyperthermia, nanoparticle-wrapped cisplatin chemotherapy, and magnetic particles of albumin. In addition, to combine the multiple-killing effects of hyperthermal targeting therapy, chemotherapy, and radiotherapy, the albumin-nanoparticle surfaces were linked with radionuclide (188)Re-labeled folic acid ligand ((188)Re-folate-CDDP/HSA). METHODS: Human serum albumin was labeled with (188)Re using the pre-tin method. Reaction time and optimal conditions of labeling were investigated. The particles were intravenously injected into mice, which were sacrificed at different time points. Radioactivity per gram of tissue of percent injected dose (% ID/g) was measured in vital organs. The biodistribution of (188)Re-folate-CDDP/HAS magnetic nanoparticles was assessed. RESULTS: Optimal conditions for (188)Re-labeled folate-conjugated albumin combined with cisplatin magnetic nanoparticles were: 0.1 mL of sodium gluconate solution (0.3 mol/L), 0.1 mL of concentrated hydrochloric acid with dissolved stannous chloride (10 mg/mL), 0.04 mL of acetic acid buffer solution (pH 5, 0.2 mol/L), 30 mg of folate-conjugated albumin combined with cisplatin magnetic nanoparticles, and (188)ReO(4) eluent (0.1 mL). The rate of (188)Re-folate-CDDP-HSA magnetic nanoparticle formation exceeded 90%, and radiochemical purity exceeded 95%. The overall labeling rate was 83% in calf serum at 37°C. The major uptake tissues were the liver, kidney, intestine, and tumor after the (188)Re-folate-CDDP/HSA magnetic nanoparticles were injected into nude mice. Uptake of (188)Re-folate-CDDP/HSA magnetic nanoparticles increased gradually after injection, peaked at 8 hours with a value of 8.83 ± 1.71, and slowly decreased over 24 hours in vivo. CONCLUSION: These results indicate that (188)Re-folate-CDDP/HSA magnetic nanoparticles can be used in radionuclide-targeted cancer therapy. Surface-modified albumin nanoparticles with folic acid ligand-labeled radionuclide ((188)Re) were successfully prepared, laying the foundation for a triple-killing effect of thermotherapy, chemotherapy, and radiation therapy.

Phosphorylated p27Kip1 on Thr157 is an important prognosis in human hepatocellular carcinoma in vivo and in vitro.

The p27(Kip1) cyclin-dependent kinase inhibitor is a negative regulator of cell cycle progression in G(1) phase; recent studies suggested that oncogenically activated kinase Akt/PKB can also phosphorylate p27(kip1) at T157 inducing its relocalization to the cytoplasm. To evaluate the significance of p-p27 Thr157 and PI3K pathway in hepatocellular carcinoma (HCC), we studied 51 hepatocellular carcinomas along with corresponding nontumoral tissue and the HCC cell lines. Immunohistochemistry and western blot analysis suggested that p-p27 Thr157 was overexpressed in HCC, which was positively correlated with proliferation marker Ki-67. Correlation analysis was performed among immunohistochemistry-assessed level of p-p27 Thr157, survival, and major clinical and pathological variables. Overexpressed p-p27 Thr157 was correlated with histological differentiation (P < 0.05). Univariate analysis showed that p-p27 Thr157 and Ki-67 expression were correlated with tumor-specific survival. In a multivariate analysis, p-p27 Thr157 and Ki-67 protein expression were proved to be an independent prognostic for HCC. While in vitro, treatment of LY294002 and transduction of mutant p27 (T157A) could diminish the expression of p-p27 Thr157 protein and arrest cells growth. Our results suggested that p-p27 Thr157 protein expression may be a favorable independent poor prognostic parameter for HCC. Gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K and transduction of mutant p27 (T157A) to down-regulate p-p27 Thr157 expression could be developed for the management of HCC.

The preparation and characterization of folate-conjugated human serum albumin magnetic cisplatin nanoparticles.

OBJECTIVE: Nanoparticles are becoming an important method of targeted drug delivery. To evaluate the importance of folate-conjugated human serum albumin (HSA) magnetic nanoparticles (Folate-CDDP/HSA MNP), we prepared drug-loaded Folate-CDDP/HSA MNPs and characterized their features. METHODS: First, folate was conjugated with HSA under the effect of a condensing agent, and the conjugating rate was evaluated by a colorimetric method using 2, 4, 6 - trinitrobenzene sulfonic acid. Second, under N2 gas, Fe3O4 magnetic nanomaterials were prepared and characterized by using transmission electron microscopy (TEM), SEM-EDS and X-ray diffraction (XRD). Finally, Folate-CDDP/HSA MNP was prepared by using a solvent evaporation technique. TEM was used to observe particle morphology. The particle size and distribution of the prepared complexes were determined by a Laser particle size analyzer. Drug loading volume and drug release were investigated by a high performance liquid chromatography method (HPLC) in vitro. RESULTS: We successfully prepared folate-conjugated HSA and its conjugating rate was 27.26 µg/mg. Under TEM, Fe3O4 magnetic nanoparticles were highly electron density and had an even size distribution in the range of 10-20 nm. It was confirmed by SEM-EDS and XRD that Fe3O4 magnetic nanoparticles had been successfully prepared. Under TEM, drug-loaded magnetic nanoparticles were observed, which had a round shape, similar uniform size and smooth surface. Their average size was 79 nm which was determined by laser scattering, and they exhibited magnetic responsiveness. Encapsulation efficiency was 89.75% and effective drug loading was calculated to be 15.25%. The release results in vitro showed that the half release time (t½) of cisplatin in cisplatin Solution and Folate-CDDP/HSA MNP was 65 min and 24 h respectively, which indicated that microspheres had an obvious effect of sustained-release. CONCLUSION: Folate-CDDP/HSA MNPs were prepared successfully. The preparation process and related characteristics data provided a foundation for further study, including the mechanism of the nanoparticles distribution in vivo and their intake by tumor cells.

The expression and prognosis of FOXO3a and Skp2 in human hepatocellular carcinoma.

The forkhead box proteins (FOXO proteins) comprise a large family of functionally diverse transcription factors involved in cellular proliferation, transformation, differentiation and longevity. Recently, ubiquitination and proteasome degradation of FOXO3a have been reported. In this study, we investigated the role of FOXO3a and Skp2 in human hepatocellular carcinoma progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 91 specimens. Furthermore in vitro, western-blot analysis and protein stabilization studies were used to study the relationship between FOXO3a and Skp2. We found that the expression of FOXO3a was negatively related with Skp2 expression (r = -0.583; p < 0.05) and FOXO3a expression correlated significantly with histological grade (p = 0.000), cirrhosis (p = 0.015), and tumor size (p = 0.043) while Skp2 expression correlated significantly with histological grade (p = 0.000) and tumor size (p = 0.005). Kaplan-Meier analysis revealed that survival curves of low versus high expressers of FOXO3a and Skp2 showed a highly significant separation in HCC (p < 0.01). Our results suggested that FOXO3a and Skp2 may be considered to be important prognosis in human hepatocellular carcinoma. In vitro studies suggested that the degradation of FOXO3a may dependent on the expression of Skp2 in the proliferated Huh7 cells.

The intra-nucleus integration of mitochondrial DNA (mtDNA)in cervical mucosa cells and its relation with c-myc expression.

OBJECTIVE: To explore the relationship between the integration of mitochondrial DNA(mtDNA) in the nuclei of cervical epithelium cells and the expression of c-myc. METHODS: The expression of c-myc protein was measured by immunohistochemical test in 40 cases of the uterine cervix cancer, 30 cases of cervical intraepithelial neoplasia (CIN) and 30 cases of normal cervical epithelium; the sequence of mtDNA in the nuclei was detected by in situ hybridization technique. RESULTS: The detection rates of mtDNA in the nuclei of cervical epithelium cells were 27.5%, 13.3% and 0% in cervical carcinoma, CIN, and normal cervical epithelium respectively. The expression rate of c-myc in cervical mucoma cells was 67% in the mtDNA sequence positive group and was significantly higher than that in the negative group (36%). CONCLUSION: The integration of mtDNA into the nuclei of cervical epithelium cells may be involved in the carcinogenesis of cervical epithelium cells and the expression of c-myc might be related to the integration of mtDNA sequence into nuclei of cervical epithelium cells.

[Expression and significance of CD44v6, bcl-2, and vascular endothelial growth factor(VEGF) in endometrial adenocarcinoma].

BACKGROUND & OBJECTIVE: The pathogenesis of endometrial carcinoma has not been clear yet. The aim of this study was to investigate the influence of expression of CD44v6, bcl-2, and vascular endothelial growth factor (VEGF) on oncogenesis and progression in endometrial carcinoma. METHODS: The expression levels of CD44v6, bcl-2, and VEGF were determined using immunohistochemistry in 55 cases with endometrial adenocarcinoma, 10 cases each of normal proliferative endometrium, simple hyperplasia, and atypical hyperplasia, respectively. RESULTS: (1)The expression of CD44v6 and VEGF increased gradually from normal proliferative endometrium to simple hyperplasia, atypical hyperplasia,and adenocarcinoma, which showed highly significant difference (P< 0.001, P< 0.001), whose ratios were 10%, 40%, 60%, 78.18% and 0%, 0%, 10%, 83.64%,respectively. While the expression of bcl-2 showed no significant difference among the above different tissues. (2)The CD44v6 expression in endometrial carcinoma was inversely associated with surgical stages and lymph node metastasis(P< 0.05, P< 0.01). The bcl-2 expression was found to be significantly related to histologic grades of the tumor (P< 0.05). The VEGF expression was significantly associated with surgical stages, myometrial invasion, and lymph node status. (3)There was statistically significant correlation between bcl-2 and CD44v6, bcl-2 and VEGF expression (P< 0.05, P< 0.05). (4)The univariate analysis revealed that the expression of CD44v6 and bcl-2 were significantly related to the prognosis of the patients (P< 0.01, P< 0.05). Cox proportional hazards model analysis showed that the prognosis was independently affected by age, surgical stage, and CD44v6 expression. CONCLUSION: CD44v6, bcl-2, and VEGF play roles in oncogenesis and progression of endometrial adenocarcinoma. Detection of these gene proteins may be helpful for early diagnosis, prognosis prediction, and treatment of endometrial carcinoma.