Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Enhanced growth of lactobacilli and bioconversion of isoflavones in biotin-supplemented soymilk upon ultrasound-treatment

This study aimed at utilizing ultrasound treatment to further enhance the growth of lactobacilli and their isoflavone bioconversion activities in biotin-supplemented soymilk. Strains of lactobacilli (Lactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633, L. gasseri FTDC 8131) were treated with ultrasound (30 kHz, 100 W) at different amplitudes (20%, 60% and 100%) for 60, 120 and 180 s prior to inoculation and fermentation in biotin-soymilk. The treatment affected the fatty acids chain of the cellular membrane lipid bilayer, as shown by an increased lipid peroxidation (P < 0.05). This led to increased membrane fluidity and subsequently, membrane permeability (P < 0.05). The permeabilized cellular membranes had facilitated nutrient internalization and subsequent growth enhancement (P < 0.05). Higher amplitudes and longer durations of the treatment promoted growth of lactobacilli in soymilk, with viable counts exceeding 9 log CFU/mL. The intracellular and extracellular β-glucosidase specific activities of lactobacilli were also enhanced (P < 0.05) upon ultrasound treatment, leading to increased bioconversion of isoflavones in soymilk, particularly genistin and malonyl genistin to genistein. Results from this study show that ultrasound treatment on lactobacilli cells promotes (P < 0.05) the β-glucosidase activity of cells for the benefit of enhanced (P < 0.05) isoflavone glucosides bioconversion to bioactive aglycones in soymilk.     

In vitro activation of mitochondria-caspase signaling pathway in sonodynamic therapy-induced apoptosis in sarcoma 180 cells

Sonodynamic therapy (SDT) has been shown to mediate apoptosis in many experimental systems, but the detailed mechanism of this process is unclear. In this study, we aim to investigate the potential participation of the mitochondria-caspase signaling pathway in the SDT-induced apoptosis in isolated sarcoma 180 (S180) cells. The cell suspension was treated with 1.75 MHz continuous ultrasound (US) at an acoustic intensity (I_SATA) of 1.4 W for 3 min in the absence or presence of 20 μg/ml hematoporphyrin (Hp). At different times after the SDT-treatment, the apoptotic cells were identified under a scanning electron microscope, and the apoptosis index (AI) was determined by flow cytometry. In addition, the mitochondrial membrane potential, permeabilization of the inner mitochondrial membrane, and translocation of apoptosis-related proteins were assessed by confocal microscopy. Simultaneously, the activation of some special apoptosis-associated proteins [caspase-9, caspase-3, polypeptide poly (ADP-ribose) polymerase (PARP), and Bax] was evaluated by western blotting. Our results indicate that the ultrasonically activated Hp can cause obvious cell apoptosis (AI, 57.66%) at 3 h after treatment, and this effect can be significantly reduced by caspase-9 inhibitor (AI, 20.76%) and the oxygen scavenger NaN₃ (20.11%). However, the apoptosis induced by ultrasound alone was relatively lower (28.33%) and was not reduced by NaN₃. Further, SDT caused an 82.1% reduction in the mitochondrial membrane potential and a 70.7% reduction in the permeabilization of the inner mitochondrial membrane immediately after treatment, and these two effects were obviously prevented by NaN₃. In comparison with the control cells, the SDT-treated cells showed obvious cytochrome-c and Bax translocations, caspase activation, Bax expression, and PARP cleavage at 1 h after SDT-treatment. However, in the cells treated with ultrasound alone, these phenomena partially and weakly occurred 3 h after exposure. These results primarily showed that the mitochondria-caspase signaling pathway in S180 cells was activated in the US- and SDT-induced apoptosis. Moreover, Hp significantly accelerates the process of apoptosis and enhances the cytotoxic effect of ultrasonic treatment. Singlet oxygen may be responsible for the mitochondrial damage and the activation of the apoptotic signaling pathway.     

Enhancement of ultrasonically induced cell damage by phthalocyanines in vitro

In this work, erythrocytes from carp were used as a nucleated cell model to test the hypothesis that the phthalocyanines (zinc - ZnPc and chloroaluminium -AlClPc) enhance ultrasonically induced damage in vitro. In order to confirm and complete our earlier investigation, the influence of ultrasound (US) and phthalocyanines (Pcs) on unresearched cellular components, was studied. Red blood cells were exposed to 1 MHz continuous ultrasound wave (0.61 and/or 2.44 W/cm²) in the presence or absence of phthalocyanines (3 μM). To identify target cell damage, we studied hemolysis, membrane fluidity and morphology of erythrocytes. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric methods using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5,-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The effect of US and Pcs on nucleated erythrocytes morphology was estimated on the basis of microscopic observation.The enhancement of ultrasonically induced membrane damage by both phthalocyanines was observed in case of hemolysis, and membrane surface fluidity, in comparison to ultrasound. The authors also observed changes in the morphology of erythrocytes. The obtained results support the hypothesis that the Pcs enhance ultrasonically induced cell damage in vitro.Furthermore, the influence of ultrasound on phthalocyanines (Pcs) in medium and in cells was tested. The authors observed changes in the phthalocyanines absorption spectra in the medium and the increase in the intensity of phthalocyanines fluorescence in the cells. These data can suggest changes in the structure of phthalocyanines after ultrasound action.     

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

Objective

The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT).

Methods

The MB concentration was kept constant at 100 μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5 s with an intensity of 0.46 W/cm². The cytotoxicity was investigated 24 h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT.

Conclusions

Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.     

Sonodynamically induced anti-tumor effect with protoporphyrin IX on hepatoma-22 solid tumor

Objective

The purpose of this study was to evaluate sonodynamically induced anti-tumor effect of protoporphyrin IX (PPIX) in mice bearing hepatoma-22 (H-22) solid tumors, and the possible in vivo cell damage mechanism was also investigated.

Methods

The pharmacokinetics of PPIX was analyzed in plasma, skin, muscle and tumor of H-22 bearing mice. Tumors were irradiated with ultrasound (1.43 MHz, I_SATA 3 W/cm², 3 min) for three times at 8, 12 and 24 h after 5.0 mg/kg PPIX administration, respectively. The anti-tumor effects of sonodynamic therapy (SDT) were estimated by the tumor inhibition ratio (volume and weight). The bio-effects of SDT were evaluated by hematoxylin and eosin (H&E) staining, transmission electron microscope (TEM) observation, lipid peroxidation (LPO) measurement and anti-oxidative enzymes (glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD)) assay.

Results

A significant anti-tumor effect was obtained by PPIX-mediated sonodynamic therapy (PPIX-SDT). At the fifteenth day after PPIX-SDT, the tumor growth and tumor weight inhibition ratios were 53.84% and 45.86%, respectively. In addition, the structure of tumor tissues and the anti-oxidative enzymes were obviously destroyed after SDT treatment.

Conclusions

A biochemical mechanism was involved in PPIX-SDT in vivo, and the free radicals produced by the synergistic treatment destroying the anti-oxidative system of tumor cells in vivo may play an important role in this action. Also, the thermal effect could not be excluded in inducing damage of cellular structures, like membrane disruption and chromatin condensation under current evaluation in this paper.     

Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells

Sonodynamic therapy (SDT) is a promising modality for cancer treatment, involving the synergistic interaction of ultrasound and some chemical compounds termed as sono-sensitizers. It has been found that SDT can lead to apoptotic cell death because of the induction of direct sonochemical and subsequent redox reactions. However, the detailed mechanisms are not clear. This study was to identify the cytotoxic effects of ultrasound-activated protoporphyrin IX (PpIX) on MDA-MB-231 cells. The fluorescence microscope was used to detect the sub-cellular localization of PpIX. Several distinct sonochemical effects were found after SDT treatment, including the decrease of cell viability, generation of intracellular ROS, the loss of mitochondrial membrane potential. The activation of some special apoptosis-associated proteins [Caspase-9, Caspase-3 and polypeptide poly (ADP-robose) polymerase] was evaluated by western blotting. The results show that PpIX mediated SDT (PpIX-SDT) treatment could obviously inhibit the proliferation of MDA-MB-231 cells, and which was significantly reduced by the pan-Caspase inhibitor z-VAD-fmk and the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC). Further, SDT induced a conspicuous loss of mitochondrial membrane potential (MMP) and a mass of ROS accumulation in MDA-MB-231 cells at 1 h post-treatment and the SDT-treated cells showed obvious Caspase-3 and Caspase-9 activation, and PARP cleavage at 6 h after treatment. And, the general apoptosis marker-Caspase-3 activation-was also greatly relieved by NAC. These findings primarily indicate a Caspase-depended apoptosis could be induced by PpIX-SDT in MDA-MB-231 cells, and the intracellular ROS was involved during the apoptotic process.     

Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells

Objectives

The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT).

Methods

The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (4′-6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis.

Results

The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 μg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase-3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes.

Conclusions

These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis.     

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

Objectives

Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.

Methods

Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 μm curcumin and then were treated by ultrasound for 8 s at the intensity of 0.46 W/cm². Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM).

Results

MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together. The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2 cells after the combined treatment of curcumin and ultrasound.

Conclusions

Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.     

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity I_SATA (spatial averaged, temporal averaged) varied from 0.33 W/cm² to 8 W/cm² (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm².     

Surface characterization of Pd/Ag23 wt% membranes after different thermal treatments

Hydrogen permeation measurements of 1.5-10 μm thick Pd/Ag23 wt% membranes before and after thermal treatments at 300 °C in air (both sides) or in the temperature range 300-450 °C in N₂ (feed side) and Ar (permeate side) were performed. Accompanying changes in surface topography and chemical composition were subsequently investigated by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) depth profiling. For a 2 μm thick membrane, the surface roughness increased for all annealing temperatures applied, while a temperature of 450 °C was required for an increase in roughness of both membrane surfaces to occur for the 5 μm membrane. The thickest membrane, of 10 μm, showed changed surface roughness on one side of the membrane only and a slight decrease in hydrogen permeance after all heat treatments in N₂/Ar. X-ray photoelectron spectroscopy investigations performed after treatment and subsequent permeation measurements revealed segregation of silver to the membrane surfaces for all annealing temperatures applied. In comparison, heat treatment at 300 °C in air resulted in significantly increased hydrogen permeance accompanied by increasing surface roughness. Upon exposure to oxygen, Pd segregates to the surface to form a 2-3 nm thick oxide layer (PdO), with more complex segregation behavior after subsequent reduction and permeance measurements in pure hydrogen. The available permeance data for the Pd/Ag23 wt% membranes after heat treatment in air at 300 °C is found to depend linearly on the inverse membrane thickness, implying bulk limited hydrogen permeation for thicknesses down to 1.5-2.0 μm.     

Sonodynamic inactivation of methicillin-resistant Staphylococcus aureus in planktonic condition by curcumin under ultrasound sonication

Methicillin-Resistant Staphylococcus aureus (MRSA) is an important cause of difficult-to-treat infections. The present study aims to investigate sonodynamic inactivation of MRSA in planktonic condition using curcumin under ultrasound sonication. Dark toxicity of curcumin to MRSA was investigated to choose the concentration range of curcumin used in the study. The uptake of curcumin in MRSA was observed before ultrasound sonication. After sonication colony forming units (CFUs) and bacterial viability were investigated using fluorescence assay. Additionally, chromosomal DNA fragmentation was also analyzed. Curcumin showed no dark toxicity to MRSA in the concentration range of ?500 μM. The maximum uptake of curcumin in MRSA occurred in 50 min after curcumin incubation. Counting of CFUs showed that curcumin had significantly sonodynamic killing effect on MRSA in a curcumin dose-dependent manner, and 5-log reduction in CFU was observed after curcumin treatment (40 μM) at room temperature in the dark for 50 min followed by exposure to ultrasound with intensity of 1.56 W/cm² for 5 min. The ratio of green-fluorescent intensity to red-fluorescent intensity was obviously decreased after curcumin treatment under ultrasound sonication. No significant change in chromosomal DNA was found in the cultured MRSA after the combined treatment of curcumin and ultrasound. These results demonstrated that sonodynamic action of curcumin had significant inactivation of MRSA in planktonic condition.     

Ultrasound-assisted conversion of alpha-chitin into chitosan

This paper discusses the production of chitosan by applying high intensity ultrasound irradiation to alpha-chitin suspended in 40% aqueous sodium hydroxide. The average degree of acetylation (DA) of chitosan was determined by ¹H NMR spectroscopy and titrimetry while its viscosity average molecular weight (Mv) was calculated from the intrinsic viscosity as determined by capillary viscometry. The results show that fully acid-soluble chitosans (DA < 32%; 100,000 g/mol ? Mv ? 200,000 g/mol) are produced at very high yield (>95%) by applying non-isothermal ultrasound-assisted N-deacetylation process to alpha-chitin suspension (44 mg/mL). It is also shown that such a process is more efficient than thermochemical N-deacetylation, even being carried out at a lower temperature due to the effects of high intensity ultrasound irradiation.     

Ultrasound treatment enhances cholesterol removal ability of lactobacilli

This study aimed to evaluate the effect of ultrasound treatment on the cholesterol removing ability of lactobacilli. Viability of lactobacilli cells was significantly increased (P < 0.05) immediately after treatment, but higher intensity of 100 W and longer duration of 3 min was detrimental on cellular viability (P < 0.05). This was attributed to the disruption of membrane lipid bilayer, cell lysis and membrane lipid peroxidation upon ultrasound treatment at higher intensity and duration. Nevertheless, the effect of ultrasound on membrane properties was reversible, as the viability of ultrasound-treated lactobacilli was increased (P < 0.05) after fermentation at 37 °C for 20 h. The removal of cholesterol by ultrasound-treated lactobacilli via assimilation and incorporation of cholesterol into the cellular membrane also increased significantly (P < 0.05) upon treatment, as observed from the increased ratio of membrane C:P. Results from fluorescence anisotropies showed that most of the incorporated cholesterol was saturated in the regions of phospholipids tails, upper phospholipids, and polar heads of the membrane bilayer.     

Biological effects of combined ultrasound and cisplatin treatment on ovarian carcinoma cells

The effects of low-power ultrasound, the anti-cancer drug cisplatin, and their combined application were studied in two lines of human ovarian carcinoma cells, A2780 and A2780cis. Four modes of treatment were used: exposure to ultrasonic field, application of cisplatin, exposure to ultrasound followed by cisplatin, and presence of cisplatin followed by exposure to application ultrasound. Ultrasound was used at intensities of 0.5 W/cm² and 1.0 W/cm² for 10 min, cisplatin was applied at concentrations of 1 μM and 6 μM per cell suspension treated in A2780 and cisplatin-resistant A2780cis cells, respectively. The results of each experimental treatment were assessed by the resultant cell viability related to the viability of control cells, using a standard MTT test. It was shown that a combined effect of ultrasound and cisplatin was more effective than that of ultrasound or cisplatin alone. It also appeared that the order of application played a role, with the cisplatin-ultrasound treatment lowering cell viability more than the ultrasound-cisplatin treatment. It can be assumed that the exposure of cells to a low-power ultrasonic field has an immediate effect on the structure of cell surfaces and, consequently, on entry of cisplatin into the cell.The study also included observations on changes in the cell cycle associated with the treatments used in both cell lines and their evaluation by flow cytometry.     

Instant magnetic labeling of tumor cells by ultrasound in vitro

Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.     

Study on the interaction of tropisetron hydrochloride and l-tryptophan by spectrofluorimetry and its analytical application

A new method to determine tropisetron hydrochloride with l-tryptophan in the medium with pH=9.0 was studied, which is based on the fluorescence quenching effect of tropisetron hydrochloride on l-tryptophan. The fluorescence quenching mechanism and various factors influencing fluorescence quenching were discussed. Under the optimum conditions, the linear range and detection limit were 0.03-12.0 and 0.01 μg/mL (correlation coefficient r=0.9970), respectively. The calibration curve equation was ΔF=6.17+12.56 C (μg/mL). RSD was 3.4% (c=4.0 μg/mL, n=5); the detection limit estimated (S/N=3) was 0.01 μg/mL. The proposed method had been successfully applied to determine tropisetron hydrochloride in real samples and the obtained results were in good agreement with the results of the official method.     

Preparation and in vitro evaluation of poly(D,L-lactide-co-glycolide) air-filled nanocapsules as a contrast agent for ultrasound imaging

The aim of this study was to prepare air-filled nanocapsules intended ultrasound contrast agents (UCAs) with a biodegradable polymeric shell composed of poly(d,l-lactide-co-glycolide) (PLGA). Because of their size, current commercial UCAs are not capable of penetrating the irregular vasculature that feeds growing tumors. The new generation of UCAs should be designed on the nanoscale to enhance tumor detection, in addition, the polymeric shell in contrast with monomolecular stabilized UCAs improves the mechanical properties against ultrasound pressure and lack of stability. The preparation method of air-filled nanocapsules was based on a modification of the double-emulsion solvent evaporation technique. Air-filled nanocapsules with a mean diameter of 370 ± 96 nm were obtained. Electronic microscopies revealed spherical-shaped particles with smooth surfaces and a capsular morphology, with a shell thickness of ∼50 nm. Air-filled nanocapsules showed echogenic power in vitro, providing an enhancement of up to 15 dB at a concentration of 0.045 mg/mL at a frequency of 10 MHz. Loss of signal for air-filled nanocapsules was 2 dB after 30 min, suggesting high stability. The prepared contrast agent in this work has the potential to be used in ultrasound imaging.