Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil

BACKGROUND: Super-paramagnetic iron oxide nanoparticles (SPION) contain a chemotherapeutic drug and are regarded as a promising technique for improving targeted delivery into cancer cells. RESULTS: In this study, the fabrication of 5-fluorouracil (5-FU) was investigated with loaded Dextran (DEXSPION) using the co-precipitation technique and conjugated by folate (FA). These nanoparticles (NPs) were employed as carriers and anticancer compounds against liver cancer cells in vitro. Structural, magnetic, morphological characterization, size, and drug loading activities of the obtained FA-DEX-5-FUSPION NPs were checked using FTIR, VSM, FESEM, TEM, DLS, and zeta potential techniques. The cellular toxicity effect of FA-DEX-5-FU-SPION NPs was evaluated using the MTT test on liver cancer (SNU-423) and healthy cells (LO2). Furthermore, the apoptosis measurement and the expression levels of NF-1, Her-2/neu, c-Raf-1, and Wnt-1 genes were evaluated post-treatment using flow cytometry and RT-PCR, respectively. The obtained NPs were spherical with a suitable dispersity without noticeable aggregation. The size of the NPs, polydispersity, and zeta were 74 ± 13 nm, 0.080 and 45 mV, respectively. The results of the encapsulation efficiency of the nano-compound showed highly colloidal stability and proper drug maintenance. The results indicated that FA-DEX-5-FU-SPION demonstrated a sustained release profile of 5-FU in both phosphate and citrate buffer solutions separately, with higher cytotoxicity against SNU-423 cells than against other cells types. These findings suggest that FA-DEX-SPION NPs exert synergistic effects for targeting intracellular delivery of 5-FU, apoptosis induction, and gene expression stimulation. CONCLUSIONS: The findings proved that FA-DEX-5-FU-SPION presented remarkable antitumor properties; no adverse subsequences were revealed against normal cells.

Preclinical application of MR and fluorescent dual-modality imaging combined with photothermal therapy in HER-2 positive breast cancer / 中华肿瘤杂志

Objective@#To construct superparamagnetic iron oxide nanoparticles (SPIONs) coated on trastuzumab and indocyanine green (ICG) and then investigate whether the coated nanoparticles (NPs) targeted to human epidermal growth factor receptor-2 (HER-2) receptors on breast cancer cells in vitro and in vivo.@*Methods@#The Fe3O4-trastuzumab-ICG NPs were constructed. And a series of characteristics of the NPs were evaluated. The uptake ability of SK-BR-3, a HER-2 positive breast cancer cell, was observed by transmission electron microscopy. Then the NPs were injected in the tail veins of SK-BR-3 xenograft tumor-bearing mice to observe the aggregation of NPs in the tumor sites by MRI and fluorescent imaging. Furthermore, when the NPs was gathered at the tumor sites, the near infrared thermal imaging system was used to monitor the tumor temperature after the near infrared radiation.@*Results@#The successfully constructed Fe3O4-trastuzumab-ICG NPs had the size of (25.93±4.25) nm. The absorption peak was 828 nm, which was as same as the emission wavelength of ICG. The NPs had a high relaxation rate of approximately 107.65 mM-1·s-1. The maximum temperature of NPs solution could reach to 57.8℃ after continuous near infrared laser irradiation. The transmission electron microscopy imaging revealed that the NPs could target and enter into the endoplasmic reticulum of SK-BR-3 cells. MRI analysis showed the lowest T2 relaxation time in the tumor sites 24 h after tail vein injection of the NPs. The △T2 of the tumor sites in the Fe3O4-trastuzumab-ICG group (30.7±4.8) ms was higher compared with that of control group (3.1±1.1) ms, Fe3O4-IgG-ICG group (4.4±0.9) ms and trastuzumab + Fe3O4-trastuzumab-ICG group (11.3±3.8) ms., respectively, all showing statistically significant differences (P<0.05). The fluorescence imaging revealed that the NPs was concentrated transiently in the intraperitoneal organs and tumor sites, then excreted into the bladder. After 24 h, there was an obvious aggregation in the tumor sites. The near infrared thermal imaging experiments showed that the temperature of tumor sites in Fe3O4-trastuzumab-ICG group could go up to 49.4℃ after continuous near infrared light irradiation.@*Conclusion@#The newly constructed Fe3O4-trastuzumab-ICG NPs have the potential to act as a multifunctional imaging agent and a powerful tool for photothermal therapy for HER-2 positive breast cancer.

Preparation of PR-SPIO-PLGA molecular probe and its property / 吉林大学学报(医学版)

Objective: To explore the method to construct the progesterone receptor (PR)-superparamagnetic iron oxide (SPIO)-poly lactic-co-glycolic acid (PLGA) and perfluorohexane (PFP) molecular probe (PR-s-PFP/PLGA), and to investigate its targeted possibility of binding the breast cancer cells in vitro. Methods: The s-PFP/PLGA nanoparticles were prepared and the diameter and average potential of s-PFP/PLGA nanoparticles were detected. Cytotoxicity experiment was used to compare the relative proliferation rates (RGR) of cells with different concentrations of s-PFP/PLGA. The magnetic resonance imaging (MRI), photoacoustic imaging (PAI) and ultrasound imaging of s-PFP/PLGA nanoparticles were detected, and the echo intensity of s-PFP/PLGA nanoparticles was observed by high intensity focused ultrasound (HIFU) invitro. The PR-s-PFP/PLGA probe was prepared by carbodiimide method. The breast cancer cell line T-47d with high expression of PR was cultured in vitro. The T-47d cells with DiO green fluorescence were divided into targeted imaging agent group, non-targeted group and blank control group. The binding of the probe to the cells in various groups was observed. Results: The s-PFP/PLGA nanoparticles were spherical under transmission electron microscope (TEM). The SPIO particles were evenly distributed on the shell, the average particle size was (738. 5 + 181. 2) nm and the average potential was (-15. 8 + 5. 7) mV, and the obvious photoacoustic signals were found. The ultrasound imaging of s-PFP/PLGA nanoparticles in vitro showed a punctate high-level echo, and the echo intensity was enhanced after HIFU irradiation. With the increasing of iron concentrations, the MRI signals showed a increasing trend in T1W1. Under laser scanning confocal microscope, the s-PFP/PLGA nanoparticles, which was phagocytized by T-47d cells, emitted red fluorescence. Conclusion; PR-SPIO-PLGA has excellent physicochemical properties, good stability and strong targeting effect on the cancer cells.

Magnetic resonance imaging of macrophages for the diagnosis of rat collagen-induced arthritis: a preliminary study / 中华风湿病学杂志

Objective To explore the optimal time and sequence for getting the best magnetic resonance (MR) imaging when MR image of synovium macrophages was used for the diagnosis of collageninduced arthritis (CIA) in a rat model,and whether it can be used to monitor the efficacy of drug treatment.Methods CIA was induced by subcutaneous injection of chicken type Ⅱ collagen and complete Freund's adjuvant (CFA).Arthritis rats were randomly divided into the model group,the leflunomide group and the control group.Knees of the model group rats were imaged before and 24 h,48 h,72 h after USPIO intravenous administration (300 μmol Fe/kg) on day 28,29,30,31,respectively.From day 28,the leflunomide group was given a gauge of drug at a dose of 8 mg/kg.Then they were imaged before and 24 hours after USPIO administration on day 42,43 respectively.MR sequences included SE T1WI,SE T2WI,GRE T2 * WI.After the completion of MR imaging,rats were sacrificed to obtain histopathologic samples of synovial membrane.LSD-t test and one-way analysis of variance (ANOVA) were used for statistical analysis.Results No distinct signal enhancements were observed on the 24 h,48 h,72 h post-contrast enhancement on T1WI.On T2WI,signal intensity ratio of synovium (SNR) pre-contrast and 24 h,48 h post-contrast were 24.13±1.96,17.09± 1.23,19.14±0.91,respectively.On T2 * WI,SNR pre-contrast and 24 h,48 h post-contrast were 22.28±0.92,11.40±0.53,17.18±0.63,respectively.Distinct signal changes were observed on 24 h,48 h post-contrast on T2WI and T2 * WI (P＜0.05).The changes between SNR at 24 h,48 h,72 h post-contrast and pre-contrast were-29.09±2.42,-20.83±2.90,-6.2±2.9 respectively on T2WI,which were-48.4±1.3,-22.9±0.8,-8.2±1.6 respectively on T2 * WI.Changes were more obvious at 24 h post-contrast than 48 h post-contrast on both T2WI and T2 * WI (P＜0.05).The quantitative analyses were coinci-dent with the visual differences in signal changes between pre-contrast and post-contrast images.Difference between △SNR of leflunomide group and the control group on T1WI was not significant,while that on T2WI and T2 * WI were significantly different (P＜ 0.01).Histological examination confirmed the uptake of iron in the macrophages of arthritic knees.Signal intensity changed more on GRE T2 * WI than SE T2WI in all arthritis rats.Conclusion GRE T2 * WI is more sensitive for the diagnose of rat CIA,and 24 h post-contrast is better than 48 h and 72h post-contrast to get better images.We successfully observed the effects of leflunomide through signal changes of synovium,and the technique maybe contribute to diagnosis and therapeutic monito-ring of rheumatoid arthritis.

Study on MR tracking of magnetically labeled swine mesenchymal stem cells after autologous transplantation into acute injured liver in vivo / 中华肝胆外科杂志

Objective To evaluate in vivo tracking of swine mesenchymal stem cells (MSCs) la-beled with super paramagnetic iron oxide (SPIO) in intraportal transplantation by a clinical 1.5T MR.Methods MSCs were isolated from swine and cultured as well as expanded, which were then incuba-ted with SPIO (Feridex I. V.). Prussian blue staining was performed for showing intracelluar irons.To establish a swine model of acute liver necrosis, 0.5 g/kg of D-galactosamine was administrated to 10 pigs. MSCs(labeled cells in six, unlabeled cells in four)were injected into liver via portal veins. MR imaging was performed with a clinical 1.5T MR immediately before and at 6 h, 3 d, 7 d, 14 d after transplantation, respectively. Results Prussian blue staining of SPIO labeled MSCs could be effec-tively labeled and the labeling efficiency was almost 100%. Signal intensity loss in liver by SPIO labe-ling on FFE sequence persisted until 14 days after transplantation. Histological analysis by Prussian blue staining showed homing of labeled MSCs in liver after 14 days, primarily distributing in hepatic sinusoids and liver parenchyma. Conclusion MSCs can be labeled with SPIO in vitro successfully.MRI can monitor magnetically labeled MSCs transplanted into liver.

A study on labeling efficiency,cellular viability and in vitro MRI of human fibroblast cells labeled with SPIO of different concentration / 重庆医科大学学报

Objective:To explore the efficiency of super-paramagnetic iron oxide(SPIO)of different concentrations in vitro labeling human fibroblast cells as well as the influence on cellular viability,and to investigate the characteristics of magnetic resonance imaging.Methods:Respectively mix pure SPIO(concentration of iron:200 ?g/ml) and SPIO-PLL compound of different concentrations with substrate(final concentrations of iron:150,100,50,25 ?g/ml;final concentration of PLL:7.5 ?g/ml).Then incubate it with Hfbs for 12,24,48 h and 72 h.Collect the cells.Assess cellular viability with Trypan Blue Dye Exclusion Method;test iron particles distribution in cells with Prussian blue Stain;observe the positions of iron particles with optical microscope and TEM and calculate the labeling rate of SPIO;perform MR scanning to measure the signal intensity change of the cells.Results:Labeling efficiency of SPIO-PLL compound is much higher than that of pure SPIO(P0.05).Prussian blue Stain test shows there are more or less blue dyed iron particles inside cytoplasm of each labeled cells.And those iron particles concentrate in endosome and lysosome(with electron microscope).Culture solution of concentration in the range of 25～50 ?g Fe/ml is safe for labeling stem cells with SPIO,and 95%～100% of the cells will be effectively labeled in 18～24 h.And the MR scanning shows obvious reduction of signal intensity of the labeled cells.Conclusion:Human fibroblast cells can be easily and safely labeled with self-made SPIO-PLL compound.Labeling with appropriate solution(iron content of 25 ?g/ml) is highly efficient and has little influence on cellular viability,furthermore,with SPIO labeling,the signal intensity of cells scanned with MR is dramatically reduced.