Targeted and ultrasound-triggered drug delivery using liposomes co-modified with cancer cell-targeting aptamers and a thermosensitive polymer

In this study, we demonstrated the feasibility of targeted and ultrasound-triggered drug delivery using liposomes co-modified with single stranded DNA aptamers that recognized platelet-derived growth factor receptors (PDGFRs) as targeting ligands for breast cancer cells and poly(NIPMAM-co-NIPAM) as the thermosensitive polymer (TSP) to sensitize these liposomes to high temperature. TSP-modified liposomes (TSP liposomes) released encapsulated calcein under 1 MHz ultrasound irradiation for 30 s at 0.5 W/cm² as well as the case under incubation for 5 min at 42 °C. Ultrasound-triggered calcein release from TSP liposomes was due to an increased local temperature, resulting from cavitation bubble collapse induced by ultrasound, and not due to an increase in the bulk medium temperature. Liposomes modified with PDGFR aptamers (APT liposomes) bound to MDA-MB-231 human breast cancer cells through PDGFR aptamers; however, they did not bind to primary human mammary epithelial cells (HMECs). The binding of APT liposomes was greatest for MDA-MB-231 cells, followed by MCF-7, WiDr, and HepG2 cancer cells. In a cell injury assay using doxorubicin (DOX)-loaded APT/TSP liposomes and ultrasound irradiation, cell viability of MDA-MB-231 at 24 h after ultrasound irradiation (1 MHz for 30 s at 0.5 W/cm²) with DOX-loaded APT/TSP liposomes was 60%, which was lower than that with ultrasound irradiation and DOX-loaded TSP liposomes or with DOX-loaded APT/TSP liposomes alone.     

Enhanced OH radical generation by dual-frequency ultrasound with TiO2 nanoparticles: Its application to targeted sonodynamic therapy

The present study demonstrated the enhanced hydroxyl (OH) radical generation by combined use of dual-frequency (0.5 MHz and 1 MHz) ultrasound (US) and titanium dioxide (TiO₂) nanoparticles (NPs) as sonocatalyst. The OH radical generation became the maximum, when 0.5 MHz US was irradiated at an intensity of 0.8 W/cm² and 1 MHz US was irradiated at intensities at 0.4 W/cm² in the presence of TiO₂ NPs under the examined conditions. After incorporation of TiO₂ NPs modified with targeting protein pre-S1/S2, HepG2 cancer cells were subjected to the dual-frequency US at optimum irradiation intensities (“targeted-TiO₂/dual-US treatment”). Growth of the HepG2 cells was reduced by 46% compared with the control condition after irradiation of dual-frequency US for 60 s with TiO₂ NPs incorporation. In contrast, HepG2 cell growth was almost the same as that in the control condition when cells were irradiated with either 0.5 MHz or 1 MHz ultrasound alone without TiO₂ NP incorporation.     

Targeted and ultrasound-triggered cancer cell injury using perfluorocarbon emulsion-loaded liposomes endowed with cancer cell-targeting and fusogenic capabilities

This study investigated the targeting and ultrasound-triggered injury of cancer cells using anticancer drug-free liposomes that contained an emulsion of perfluoropentane (ePFC5) and were co-modified with avidin as a targeting ligand for cancer cells and the hemagglutinating virus of Japan (HVJ) envelope to promote liposome fusion with the cells. These liposomes are designated as ePFC5-loaded avidin/HVJ liposomes. ePFC5-loaded liposomes were sensitized to ultrasound irradiation. Liposomes modified with avidin alone (avidin liposomes) showed binding to MCF-7 human breast cancer cells, and liposomes modified with HVJ envelope alone (HVJ liposomes) were found to fuse with MCF-7 cells. The irradiation of MCF-7 cells with 1 MHz ultrasound (30 s, 1.2 W/cm², duty ratio 30%) combined with ePFC5-loaded avidin/HVJ liposomes resulted in a decrease in cell viability at 1 h after irradiation to 43% of that of controls without ultrasound irradiation or liposomes. The cell viability was lower than that of cells treated with ultrasound irradiation with ePFC5-loaded avidin liposomes or ePFC5-loaded HVJ liposomes. This indicates that co-modification of liposome with avidin and HVJ envelope could enhance ultrasound-induced cell injury in the presence of ePFC5-loaded liposomes.     

Sonocatalytic injury of cancer cells attached on the surface of a nickel–titanium dioxide alloy plate

The present study demonstrates ultrasound-induced cell injury using a nickel–titanium dioxide (Ni–TiO₂) alloy plate as a sonocatalyst and a cell culture surface. Ultrasound irradiation of cell-free Ni–TiO₂ alloy plates with 1 MHz ultrasound at 0.5 W/cm² for 30 s led to an increased generation of hydroxyl (OH) radicals compared to nickel–titanium (Ni–Ti) control alloy plates with and without ultrasound irradiation. When human breast cancer cells (MCF-7 cells) cultured on the Ni–TiO₂ alloy plates were irradiated with 1 MHz ultrasound at 0.5 W/cm² for 30 s and then incubated for 48 h, cell density on the alloy plate was reduced to approximately 50% of the controls on the Ni–Ti alloy plates with and without ultrasound irradiation. These results indicate the injury of MCF-7 cells following sonocatalytic OH radical generation by Ni–TiO₂. Further experiments demonstrated cell shrinkage and chromatin condensation after ultrasound irradiation of MCF-7 cells attached on the Ni–TiO₂ alloy plates, indicating induction of apoptosis.     

Esterification of sodium 4-hydroxybenzoate by ultrasound-assisted solid–liquid phase-transfer catalysis using dual-site phase-transfer catalyst

The catalytic esterification of sodium 4-hydroxybenzoate with benzyl bromide by ultrasound-assisted solid–liquid phase-transfer catalysis (U-SLPTC) was investigated using the novel dual-site phase-transfer catalyst 4,4′-bis(tributylammoniomethyl)-1,1′-biphenyl dichloride (BTBAMBC), which was synthesized from the reaction of 4,4′-bis(chloromethyl)-1,1′-biphenyl and tributylamine. Without catalyst and in the absence of water, the product yield at 60 °C was only 0.36% in 30 min of reaction even under ultrasound irradiation (28 kHz/300 W) and 250 rpm of stirring speed. When 1 cm³ of water and 0.5 mmol of BTBAMBC were added, the yield increased to 84.3%. The catalytic intermediate 4,4′-bis(tributylammoniomethyl)-1,1′-biphenyl di-4-hydroxybenzoate was also synthesized to verify the intrinsic reaction which was mainly conducted in the quasi-aqueous phase locating between solid and organic phases. Pseudo-first-order kinetic equation was used to correlate the overall reaction, and the apparent rate coefficient with ultrasound (28 kHz/300 W) was 0.1057 min⁻¹, with 88% higher than that (0.0563 min⁻¹) without ultrasound. The esterification under ultrasonic irradiation using BTBAMBC by solid–liquid phase-transfer catalysis was developed.     

Experimental study on the influence of low-frequency and low-intensity ultrasound on the permeability of the Mycobacterium smegmatis cytoderm and potentiation with levofloxacin

Tuberculosis is an infectious disease caused by the bacterium M. tuberculosis. The aim of this study was to investigate the bactericidal effect and underlying mechanisms of low-frequency and low-intensity ultrasound combined with levofloxacin treatment against M. smegmatis (a surrogate of M. tuberculosis). As part of this study, M. smegmatis was continuously irradiated with low frequency ultrasound (42 kHz) using several different doses whereby both intensity (0.138, 0.190 and 0.329 W/cm²) and exposure time (5, 15 and 20 min) were varied. Flow cytometric analyses revealed that the permeability of M. smegmatis increased following ultrasound exposure. The survival rate, structure and morphology of bacteria in the lower-dose (I_SATA = 0.138 W/cm² for 5 min) ultrasound group displayed no significant differences upon comparison with the untreated group. However, the survival rate of bacteria was significantly reduced and the bacterial structure was damaged in the higher-dose (I_SATA = 0.329 W/cm² for 20 min) ultrasound group. Ultrasound irradiation (0.138 W/cm²) was subsequently applied to M. smegmatis in combination with levofloxacin treatment for 5 min. The results demonstrated that the bactericidal effect of ultrasonic irradiation combined with levofloxacin is higher compared to ultrasound alone or levofloxacin alone.     

Low-intensity pulsed ultrasound enhances cell killing induced by X-irradiation

To determine the effect of pulsed ultrasound (US) on radiation-induced cell killing, U937 and Molt-4 cell lines were exposed to 1.0 MHz US with 50% of duty factor at 0.3 W/cm² and pulsed at 1 Hz immediately after exposure to X-rays at 0, 0.5, 2.5 and 5 Gy. The cells were assayed 24 h after the treatments. The result showed significant enhancement of cell killing in the combined treatments. However, the ratio of apoptotic cells induced either by X-rays or US alone did not significantly change. These findings suggest that pulsed US can enhance the anticancer effect of X-irradiation due to US streaming under non-inertial cavitational condition. This combined treatment can potentially enhance the therapeutic effect of radiation therapy.     

Combination of therapeutic ultrasound with antibiotics interfere with the growth of bacterial culture that colonizes skin ulcers: An in-vitro study

Staphylococcus aureus and Escherichia coli are among the major bacterial species that colonize skin ulcers. Therapeutic ultrasound (TUS) produces biophysical effects that are relevant to wound healing; however, its application over a contaminated injury is not evidence-based. The objective of this research was to analyze the effect of TUS on in vitro-isolated S. aureus and E. coli, including the combination of ultrasound and antibiotics, in order to assess their antibiotic action on bacterial susceptibility. For the experiments, the bacterial strains were suspended in saline, then diluted (10⁴ CFU/mL) for irradiation (at 1 and 3 MHz, 0.5 and 0.8 W/cm² for 0 and 15 min) and the combination treatment of ultrasonication and antibiotics was administered by adding nalidixic acid (S. aureus) and tetracycline (E. coli) at concentrations equivalent to 50% of the minimum inhibitory concentration (MIC). The experiments were carried out in duplicate with six repetitions. The suspensions were inoculated on to Petri plates and incubated at 37 °C and the colony forming units (CFUs) were counted after 24 h. The results were subjected to the Shapiro–Wilk normality test, followed by parametric ANOVA and Tukey’s post hoc test at a significance level of 1%. The results demonstrated that the action of TUS at 1 MHz inhibited bacterial growth while at 3 MHz, bacterial growth was observed in both species. However, the synergistic combination of ultrasound and antibiotics was able to inhibit the growth of both bacteria completely after 15 min of ultrasonication. The results suggest that the action of ultrasound on S. aureus and E. coli are dependent on the oscillation frequency as well as the intensity and time of application. The combination of ultrasound with antibiotics was able to inhibit bacterial growth fully at all frequencies and doses in both species.     

Enhancement of artificial promoter activity by ultrasound-induced oxidative stress

We previously developed artificial promoters that were activated in response to X-ray irradiation. Sonication with 1.0 MHz ultrasound that causes intracellular oxidative stress was found to activate some of these promoters though to lesser degrees. The most sensitive one among these promoters showed intensity- and duration-dependent activations by sonication. In addition, its activation by sonication was attenuated when N-acetyl cysteine was present, suggesting the involvement of intracellular oxidative stress in the activation mechanism. Improved promoters for sensitivity to X-ray irradiation were also found more sensitive to sonication. The most improved one showed 6.0 fold enhancement after sonication with 1.0 MHz ultrasound at 1.0 W/cm² for 60 s. This enhancement was also attenuated with the presence of N-acetyl cysteine. When stably transfected HeLa cells with the most sensitive promoter were transplanted on to mice and sonicated, luciferase activity by the promoter increased to 1.35 fold in average though it was not statistically significant compared to control. Although gene regulation in vivo by sonication was not clear, this is the first report on artificially constructed promoters responsive to ultrasound.     

Influence of sodium alginate pretreated by ultrasound on papain properties: Activity,structure, conformation and molecular weight and distribution

The aim of the study was to investigate the impact of sodium alginate (ALG) pretreated by ultrasound on the enzyme activity, structure, conformation and molecular weight and distribution of papain. ALG solutions were pretreated with ultrasound at varying power (0.05, 0.15, 0.25, 0.35, 0.45 W/cm²), 135 kHz, 50 °C for 20 min. The maximum relative activity of papain increased by 10.53% when mixed with ALG pretreated by ultrasound at 0.25 W/cm², compared with the untreated ALG. The influence of ultrasound pretreated ALG on the conformation and secondary structure of papain were assessed by fluorescence spectroscopy and circular dichroism spectroscopy. The fluorescence spectra revealed that ultrasound pretreated ALG increased the number of tryptophan on papain surface, especially at 0.25 W/cm². It indicated that ultrasound pretreatment induced molecular unfolding, causing the exposure of more hydrophobic groups and regions from inside to the outside of the papain molecules. Furthermore, ultrasound pretreated ALG resulted in minor changes in the secondary structure of the papain. The content of α-helix was slightly increased after ultrasound pretreatment and no significant change was observed at different ultrasound powers. ALG pretreated by ultrasound enhanced the stability of the secondary structure of papain, especially at 0.25 W/cm². The free sulfhydryl (SH) content of papain was slightly increased and then decreased with the increase of ultrasonic power. The maximum content of free SH was observed at 0.25 W/cm², under which the content of the free SH increased by 6.36% compared with the untreated ALG. Dynamic light scattering showed that the effect of ultrasound treatment was mainly the homogenization of the ALG particles in the mixed dispersion. The gel permeation chromatography coupled with the multi-angle laser light scattering photometer analysis showed that the molecular weight (M_w) of papain/ALG was decreased and then increased with the ultrasonic pretreatment. Results demonstrated that the activity of immobilized papain improved by ultrasonic pretreatment was mainly caused by the variation of the conformation of papain and the effect of interactions between papain and ALG. This study is important to explain the intermolecular interactions of biopolymers and the mechanism of enzyme immobilization treated by ultrasound in improving the enzymatic activity. As expected, ALG pretreated by appropriate ultrasound is promising as a bioactive compound carrier in the field of immobilized enzyme.     

Variety of nanostructures induced by femtosecond laser pulses on surface of Au/Cr film stack

Several nanostructures were obtained after irradiation with femtosecond laser pulse (130 fs, 800 nm, 1 kHz pulse repetition frequency) on Au/Cr film stack. The influence of laser parameters such as fluence (0.5 J/cm², 1.5 J/cm², 3 J/cm²) and the number of pulse were investigated. With single pulse irradiation, the nanoline and nonoparticle were obtained for the pulse fluence of 0.5 J/cm² and 3 J/cm², respectively. The formation mechanism of those nanostructures was discussed. The results of this experiment demonstrate that different kinds of nanostructures could be formed by varying the laser parameters such as fluence and the number of pulse.     

Ultrasound assisted the preparation of 1-butoxy-4-nitrobenzene under a new multi-site phase-transfer catalyst – Kinetic study

In the present research work deals with the preparation of 1-butoxy-4-nitrobenzene was successfully carried out by 4-nitrophenol with n-butyl bromide using aqueous potassium carbonate and catalyzed by a new multi-site phase-transfer catalyst (MPTC) viz., N¹,N⁴-diethyl-N¹,N¹,N⁴,N⁴-tetraisopropylbutane-1,4-diammonium dibromide, under ultrasonic (40 kHz, 300 W) assisted organic solvent condition. The pseudo first-order kinetic equation was applied to describe the overall reaction. Under ultrasound irradiation (40 kHz, 300 W) in a batch reactor, it shows that the overall reaction greatly enhanced with ultrasound irradiation than without ultrasound. The present study provides a method to synthesize nitro aromatic ethers by ultrasound assisted liquid–liquid multi-site phase-transfer catalysis condition.     

Evaluation of correlation between chemical dosimetry and subharmonic spectrum analysis to examine the acoustic cavitation

Currently several therapeutic applications of ultrasound in cancer treatment are under progress which uses cavitation phenomena to deliver their effects. There are several methods to evaluate cavitation activity such as chemical dosimetry and measurement of subharmonic signals. In this study, the cavitation activity induced by the ultrasound irradiation on exposure parameters has been measured by terephthalic acid chemical dosimetry and subharmonic analysis. Experiments were performed in the near 1 MHz fields in the progressive wave mode and effect of duty cycles changes with 2 W/cm² intensity (I_SATA) and acoustic intensity changes in continuous mode on both fluorescence intensity and subharmonic intensity were measured. The dependence between fluorescence intensity of terephthalic acid chemical dosimetry and subharmonic intensity analysis were analyzed by Pearson correlation (p-value < 0.05). It has been shown that the subharmonic intensity and the fluorescence intensity for continuous mode is higher than for pulsing mode (p-value < 0.05). Also results show that there is a significant difference between the subharmonic intensity and the fluorescence intensity with sonication intensity (p-value < 0.05). A significant correlation between the fluorescence intensity and subharmonic intensity at different duty cycles (R = 0.997, p-value < 0.05) and different intensities (R = 0.985, p-value < 0.05) were shown. The subharmonic intensity (μW/cm²) significantly correlated with the fluorescence intensity (count) (R = 0.901; p < 0.05) and the fluorescence intensity due to chemical dosimetry could be estimated with subharmonic intensity due to subharmonic spectrum analysis. It is concluded that there is dependence between terephthalic acid chemical dosimetry and subharmonic spectrum analysis to examine the acoustic cavitation activity.     

Structural and permeability sensitivity of cells to low intensity ultrasound: Infrared and fluorescence evidence in vitro

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity I_ta < 0.1 W cm⁻²). Conventional 1 MHz and 3 MHz US devices of therapeutic relevance were employed with varying intensity and exposure time parameters. In this framework, upon cells exposure to US, structural changes at the molecular level were evaluated by infrared spectroscopy; alterations in plasma membrane permeability were monitored in terms of uptake efficiency of small cell-impermeable model drug molecules, as measured by fluorescence microscopy and flow cytometry. The results were related to the cell viability and combined with the statistical PCA analysis, confirming that NIH-3T3 cells are sensitive to therapeutic US, mainly at 1 MHz, with time-dependent increases in both efficiency of uptake, recovery of wild-type membrane permeability, and the size of molecules entering 3T3. On the contrary, the exposures from US equipment at 3 MHz show uptakes comparable with untreated samples.     

Pulsed laser induced damage in SOI material

Laser-induced damage in silicon-on-insulator (SOI) material is investigated with 1064 nm laser pulses. As the laser pulse duration is increased from 190 ps to 1.14 s, the damage threshold of SOI material decreases from 1.3×10¹⁰ to 7.7×10³ W/cm² in laser flux. It is found that the damage threshold varies inversely as the pulse duration for a short irradiation time, and is independent of pulse duration for a long irradiation time. The time dependence is in good agreement with a thermal model which well describes the thermal-induced damage in a semi-finite material irradiated by a Gaussian laser beam. The values of absorption coefficient and thermal conductivity under laser irradiation are calculated as 1.1×10³ cm^?1 and 0.18 Wcm^?1 K^?1, respectively, by fitting the model to the experimental results. These results on material damage can be used to predict the damage thresholds of SOI-based devices.     

Purification of serratiopeptidase from Serratia marcescens NRRL B 23112 using ultrasound assisted three phase partitioning

The ultrasound assisted three phase partitioning (UATPP) is a novel bioseparation method for separation and purification of biomolecules. In the present work, UATPP was investigated for the first time for purification of serratiopeptidase from Serratia marcescens NRRL B 23112. Effect of various process parameters such as ammonium sulphate saturation, t-butanol to crude extract ratio, pH, ultrasonic frequency, ultrasonic intensity, duty cycle and irradiation time were evaluated and optimized. The optimized conditions were found to be as follows: ammonium sulphate saturation 30% (w/v), pH 7.0, t-butanol to crude ratio 1:1 (v/v), ultrasound frequency 25 kHz, ultrasound intensity 0.05 W/cm², duty cycle 20% and irradiation time 5 min. The maximum purity and recovery obtained from UATPP was 9.4-fold and 96% respectively as compared to the three phase partitioning (TPP) (4.2-fold and 83%). Also the process time for UATPP was significantly reduced to 5 min from 1 h as compared to TPP. The results indicate that, UATPP is an efficient technique for the purification of serratiopeptidase with maximum purity, recovery and reduced processing time.     

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm²) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.     

Propargylation of indene-1,3-dione under a new phase-transfer catalyst combined with ultrasonication – A kinetic study

In the present study, kinetics of synthesis of 2,2-di(prop-2-ynyl)-1H-indene-1,3(2H)-dione was successfully carried out by propargylation of indene-1,3-dione with propargyl bromide using aqueous potassium hydroxide and catalyzed by a newly synthesized phase-transfer catalyst viz., N-benzyl-N-ethyl-N-isopropylpropan-2-ammonium bromide, PTC under ultrasonic (40 kHz, 300 W) assisted organic solvent condition. The pseudo first-order kinetic equation was applied to describe the overall reaction. Under ultrasound irradiation (40 kHz, 300 W) in a batch reactor, it shows that the overall reaction rate can be greatly enhanced with ultrasound irradiation than without ultrasound.     

Heat transfer enhancement using 2 MHz ultrasound

The present work focuses on possible heat transfer enhancement from a heating plate towards tap water in forced convection by means of 2 MHz ultrasound. The thermal approach allows to observe the increase of local convective heat transfer coefficients in the presence of ultrasound and to deduce a correlation between ultrasound power and Nusselt number. Heat transfer coefficient under ultrasound remains constant while heat transfer coefficient under silent conditions increases with Reynolds number from 900 up to 5000. Therefore, heat transfer enhancement factor ranges from 25% up to 90% for the same energy conditions (supplied ultrasonic power = 110 W and supplied thermal power = 450 W). In the same time cavitational activity due to 2 MHz ultrasound emission was characterized from mechanical and chemical viewpoints without significant results. At least, Particle Image Velocimetry (PIV) measurements have been performed in order to investigate hydrodynamic modifications due to the presence of 2 MHz ultrasound. It was therefore possible to propose a better understanding of heat transfer enhancement mechanism with high frequency ultrasound.