Preparation and in vivo evaluation of multifunctional 9°Y-labeled magnetic nanoparticles designed for cancer therapy.

Two different types of magnetic nanoparticles (MNPs) were synthesized in order to compare their efficiency as radioactive vectors, Fe3O4-Naked (80 ± 5 nm) and polyethylene glycol 600 diacid functionalized Fe3O4(Fe3O4-PEG600) MNPs (46 ± 0.6 nm). They were characterized based on the external morphology, size distribution, and colloidal and magnetic properties. The obtained specific power absorption value for Fe3O4-PEG600 MNPs was 200 W/g, indicated their potential in hyperthermia based cancer treatments. The labeling yield, in vitro stability and in vivo biodistribution profile of (90) Y-MNPs were compared. Both types of MNPs were (90)Y-labeled in reproducible high yield (>97%). The stability of the obtained radioactive nanoparticles was evaluated in saline and human serum media in order to optimize the formulations for in vivo use. The biodistribution in Wistar rats showed different pharmacokinetic behaviors of nanoparticles: a large fraction of both injected MNPs ended in the liver (14.58%ID/g for (90)Y-Fe3O4-Naked MNPs and 19.61%ID/g for (90)Y-Fe3O4-PEG600 MNPs) whereas minor fractions attained in other organs. The main difference between the two types of MNPs was the higher accumulation of (90)Y-Fe3O4-Naked MNPs in the lungs (12.14%ID/g vs. 2.00%ID/g for (90)Y-Fe3O4-PEG600 MNPs) due to their in vivo agglomeration. The studied radiolabeled magnetic complexes such as (90)Y-Fe3O4-PEG600 MNPs constitute a great promise for multiple diagnostic-therapeutic uses combining, for example, MRI-magnetic hyperthermia and regional radiotherapy.

90Y-labeled tin fluoride colloid as a promising therapeutic agent: preparation, characterization, and biological study in rats.

In this study, tin fluoride colloid (SnF-c) was prepared, labeled with yttrium-90 ((90)Y), and characterized with respect to its physicochemical properties and biological behavior in an animal model. Particle size of SnF-c, at constant concentration of SnF(2), was dependent on pH, concentration of sodium fluoride (NaF), temperature, and time. The particle size of SnF-c decreased with an increase in NaF concentration and a decrease in reaction mixture pH. Radiolabeling yield of (90)Y-SnF-c at higher temperature increased and it was greater than 98% for the preparation at 95 °C. The (90)Y-SnF-c demonstrated high in vitro stability both in human serum and human synovial fluid at 37 °C up to 7 days. In vivo distribution studies in healthy male Wistar rats of (90)Y-SnF-c (particles <1 µm), following intravenous administration, revealed that the localization takes place preferably in the liver. The (90)Y-SnF-c (particles >1 µm) was well retained in the synovial space for 96 h after intra-articular injection, whereas leakage of (90)Y from the joint was 1.96% over this period. Because of high labeling yield and stability, (90)Y-SnF-c might be a promising agent for radiosynovectomy or therapy of liver malignancies.

[The effect of applied cytotoxic drugs on biological behaviour of 99mTc-radiopharmaceuticals].

INTRODUCTION: This study was aimed at investigating the influence of certain cytotoxic drugs on the organ uptake of the following 9mTc-radiopharmaceuticals: 99mTc-2,3-dicarboxypropane-1, 1-diphosphonic acid, 99mTc-meso-2,3-dimercaptosuccinic acid, 9mTc-tin colloid and 99mTc-macraggregated albumin. Methotrexate sodium and cyclophosphamide were used as models to evaluate these effects. MATERIAL AND METHODS: Two groups of healthy male Wistar rats were treated separately by oral application of the drugs for 7 days. On the eighth day, each of the 99Tc-radiopharmaceuticals was applied in a separate group of treated animals. They were sacrificed at different time intervals and the radioactivity in the organs of interest was measured. The organ uptake of the 99mTc-radiopharmaceuticals in an additional control group of animals was also studied. RESULTS: The results obtained showed an alteration in the organ uptake of 99mTc-radiopharmaceuticals in animals treated with cytotoxic drugs. In the rats treated with methotrexate sodium, there was a higher uptake of 99mTc-meso-2,3-dimercaptosuccinic acid in the bones, stomach and intestine, a higher uptake of 99mTc-2,3-dicarboxypropane-1-,1-diphosphronic acid in the bones, intestine, blood and muscle, a lower uptake of 99mTc-tin colloid in the liver and a lower accumulation of 99mTc-macroaggregated albumin in the lungs. Cyclophosphamide-treated animals showed enhanced uptake of 99mTc-meso-2,3-dimercaptosuccinic acid in the kidneys, a twofold enhanced uptake of 99mTc-2,3-dicarboxypropane-1,1-diphosphronic acid in all organs except the stomach, a decreased uptake of 99mTc-tin colloid in the lungs, spleen and kidneys and a significantly decreased uptake of 99mTc-macroaggregated albumin in the lungs. CONCLUSION: These results confirm that both methotrexate sodium and cyclophosphamide may alter the organ uptake of 99mTc-radiopharmaceuticals in experimental animals.

Opposite effects of nanocrystalline fullerene (C(60)) on tumour cell growth in vitro and in vivo and a possible role of immunosupression in the cancer-promoting activity of C(60).

In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC(60)) on tumour cell growth in vitro and in vivo. NanoC(60) suspension was prepared by solvent exchange using tetrahydrofuran to dissolve C(60). In vitro, nanoC(60) caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (125I) nanoC(60) readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC(60) over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC(60) was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC(60)-injected mice. These data demonstrate that nanoC(60), in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.

Preparation and biodistribution of radiolabeled fullerene C60 nanocrystals.

The present study describes for the first time a procedure for the radiolabeling of fullerene (C(60)) nanocrystals (nanoC(60)) with Na (125)I, as well as the biodistribution of radiolabeled nanoC(60) ((125)I-nanoC(60)). The solvent exchange method with tetrahydrofuran was used to make colloidal water suspensions of radiolabeled nanoC(60) particles. The radiolabeling procedure with the addition of Na (125)I to tetrahydrofuran during dissolution of C(60) gave a higher radiochemical yield of radiolabeled nanoC(60) particles in comparison to the second option, in which Na (125)I was added after C(60) was dissolved. Using photon correlation spectroscopy and transmission electron microscopy, (125)I-nanoC(60) particles were found to have a crystalline structure and a mean diameter of 200-250 nm. The (125)I-nanoC(60) had a particularly high affinity for human serum albumin, displaying 95% binding efficiency after 1 h. Biodistribution studies of (125)I-nanoC(60) in rats indicated significant differences in tissue accumulation of (125)I-nanoC(60) and the radioactive tracer Na (125)I. The higher accumulation of radiolabeled nanoC(60) was observed in liver and spleen, while accumulation in thyroid, stomach, lungs and intestines was significantly lower in comparison to Na (125)I. In addition to being useful for testing the biological distribution of nanoC(60), the described radiolabeling procedure might have possible applications in cancer radiotherapy.

Preparation and in vivo evaluation of 90Y-meso-dimercaptosuccinic acid (90Y-DMSA) for possible therapeutic use: comparison with 99mTc-DMSA.

INTRODUCTION: The aim of this study was to find out if (90)Y could form a stabile complex with meso-2,3-dimercaptosuccinic acid (DMSA) and if (90)Y-DMSA may have potential for tumor therapy in the palliative treatment of bone metastases. METHODS: The preparing of (90)Y-DMSA was carried out by varying experimental parameters, such as ligand concentration, pH, time, and temperature of the reaction, in order to maximize the labeling yield. Analysis of the complexes enclosed the radiochemical quality control (instant thin-layer chromatography, paper chromatography, and high-performance liquid chromatography), determination of pharmacokinetical parameters as well as biodistribution study in healthy male Wistar rats. In vitro stability of the complexes was tested too. RESULTS: (90)Y-DMSA could be prepared in high yields (>95%) under optimized conditions of reaction. Stability studies in saline and human serum in vitro showed no significant release of activity from the ligand over 24 hours and 10 days, respectively. The preliminary biodistribution results in rat at 2 hours indicated that (90)Y-DMSA, at both pH levels, was significantly retained into bone. The uptake in the kidneys was lower for (90)Y-DMSA at pH 8.0 then at pH 3.0. The retention in other organs was negligible. CONCLUSIONS: (90)Y complexes could be made with ease with DMSA. (90)Y-DMSA was obtained in good yield and was found to be very stable. A promising biodistribution result of this complex pointed at potential in the palliative treatment of bone metastases.

Labeling, characterization, and in vivo localization of a new 90Y-based phosphonate chelate 2,3-dicarboxypropane-1,1-diphosphonic acid for the treatment of bone metastases: Comparison with 99mTc-DPD complex.

The goal of this investigation was to examine the possibilities for yttrium-90-labeling of the 2,3-dicarboxypropane-1,1-diphosphonic acid (DPD), which is currently labeled with technetium-99m and as a (99m)Tc-DPD clinically used as bone imaging agent. Analysis of the complex enclosed the radiochemical quality control methods, biodistribution studies, as well as the determination of pharmacokinetic parameters. The biological behavior of complexes (90)Y-DPD, (99m)Tc-DPD and (90)Y-labeled DPD-kit formulation [(90)Y-(Sn)-DPD] in animal model was compared. The labeling conditions were standardized to give the maximum yield, which ranged between 93% and 98%. The examined (90)Y complex could be easily prepared, with an outstanding yield and was also found to be very stable for at least 10h after (90)Y-labeling. Protein binding value was 4.6+/-0.7% for (90)Y-DPD complex and the complex possess a hydrophilic character. The satisfactory results of (90)Y-DPD biodistribution in healthy test animals were obtained; the uptake in the bone was 11-13%ID/g after 24h depending on the pH value during the preparation. With high skeletal uptake, a minimum uptake in soft tissues and rapid blood clearance the (90)Y-DPD complex proved to be an excellent candidate for targeting tumor therapy.

Modulation of tumor necrosis factor-mediated cell death by fullerenes.

PURPOSE: The fullerene (C60/C70 mixture-C60/70) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/nC60/70) and polyhydroxylated C60/70 [C60/70(OH)n] were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF). MATERIALS AND METHODS: TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes. RESULTS: THF/nC60/70 augmented, while C60/70(OH)n reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/nC60/70 or C60/70(OH)n. The antioxidant N-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/nC60/70 or C60/70(OH)n potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization. CONCLUSION: The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.

The mechanism of cell-damaging reactive oxygen generation by colloidal fullerenes.

Because of the ability to induce cell death in certain conditions, the fullerenes (C(60)) are potential anticancer and toxic agents. The colloidal suspension of crystalline C(60) (nano-C(60), nC(60)) is extremely toxic, but the mechanisms of its cytotoxicity are not completely understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen species (ROS)-mediated cytotoxicity of different nC(60) suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC(60)) and ethanol (EtOH/nC(60)), or by extended mixing in water (aqu/nC(60)). With regard to their capacity to generate ROS and cause mitochondrial depolarization followed by necrotic cell death, the nC(60) suspensions are ranked in the following order: THF/nC(60)>EtOH/nC(60)>aqu/nC(60). Mathematical modelling of singlet oxygen ((1)O(2)) generation indicates that the (1)O(2)-quenching power (THF/nC(60)

Multiple mechanisms underlying the anticancer action of nanocrystalline fullerene.

Using the rat glioma cell line C6 and the human glioma cell line U251, we demonstrate the multiple mechanisms underlying the in vitro anticancer effects of the C(60) fullerene water suspension (nano-C(60) or nC(60)) produced by solvent exchange method. Nano-C(60) in a dose-dependent manner reduced the tumor cell numbers after 24 h of incubation. The observed antiglioma action of nC(60) at high concentration (1 microg/ml) was due to a reactive oxygen species-mediated necrotic cell damage that was partly dependent on oxidative stress-induced activation of extracellular signal-regulated kinase (ERK). On the other hand, low-dose nC(60) (0.25 microg/ml) did not induce either necrotic or apoptotic cell death, but caused oxidative stress/ERK-independent cell cycle block in G(2)/M phase and subsequent inhibition of tumor cell proliferation. Treatment with either high-dose or low-dose nC(60) caused the appearance of acidified intracytoplasmic vesicles indicative of autophagy, but only the antiglioma effect of low-dose nC(60) was significantly attenuated by inhibiting autophagy with bafilomycin A1. Importantly, primary rat astrocytes were less sensitive than their transformed counterparts to a cytostatic action of low-dose nC(60). These data provide grounds for further development of nC(60) as an anticancer agent.

Inactivation of nanocrystalline C60 cytotoxicity by gamma-irradiation.

We investigated the effect of gamma-irradiation on the cytotoxicity of pure C60 solubilized in water by using tetrahydrofuran (THF/n-C60 or THF/n-C60). In contrast to THF/n-C60, its gamma-irradiated counterpart failed to generate oxygen radicals and cause extracellular signal-regulated kinase (ERK)-dependent necrotic cell death in various types of mammalian cells. Moreover, gamma-irradiated THF/n-C60 protected cells from the oxidative stress induced by native THF/n-C60 or hydrogen peroxide. The observed biological effects were associated with gamma-irradiation-mediated decomposition of THF and subsequent derivatization of the n-C60 surface. These results for the first time demonstrate gamma-irradiation-mediated changes in the physico-chemical properties of THF-prepared nanocrystalline C60, resulting in a complete loss of its cytotoxic effect and its conversion to a cytoprotective agent.

Distinct cytotoxic mechanisms of pristine versus hydroxylated fullerene.

The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C60) and water-soluble polyhydroxylated fullerene [C60(OH)n] were investigated. Crystal violet assay for cell viability demonstrated that nano-C60 was at least three orders of magnitude more toxic than C60(OH)n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V-fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C60 caused rapid (observable after few hours), reactive oxygen species (ROS)-associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C60(OH)n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C60 toxicity, but not C60(OH)n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C60(OH)n-induced apoptosis, but not nano-C60-mediated necrosis. Finally, C60(OH)n antagonized, while nano-C60 synergized with, the cytotoxic action of oxidative stress-inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.