2.1. Early Studies
It was chosen to include a few of the earliest papers on cellular acoustics out of historic curiosity, although the acoustic regimes of these papers are most probably beyond the inertial cavitation threshold. The articles in this section are in chronological order.
In a 1964 study, newt larvae were subjected to 5 min of 1 MHz ultrasound at intensities of 8–15 W cm
]. Their notochord cells appeared normal on light microscopy, but the endoplasmic reticulum was seen to be severely disrupted on electron microscopy. By 24 h post sonication, 50% of the endoplasmic reticulum had reverted to a normal structure.
In an early study on chromosome aberrations induced by ultrasound, two commercial foetal ultrasound devices were used [67
]. Cultured healthy-donor blood was subjected to ultrasound for 1- and 2-h durations using these devices with manufacturer specifications of 2.25 MHz and maximum power of 30 mW. The cultured cells were examined for chromosomal aberrations after sonication. There was clear evidence of substantially increased chromosomal damage in the sonicated samples compared to controls. In addition, the longer duration produced greater damage. No formal statistical significance test was reported on the results.
As an early study on bioeffects in tissue [68
], frog muscle was subjected to sonication at 85 kHz using a vibrating needle machined into a stainless steel acoustic horn. The sound amplitude was expressed in terms of the deviation of the needle tip which ranged from 1–5
m, with maximum deviation corresponding to a sub-cavitation pressure amplitude of 0.125 atm. The temperature rise was minimal and, at low amplitudes, structures such as the mitochondrial christae showed disruption. At higher amplitudes, disruption to the Z and M lines was demonstrated. Z and M lines are anatomical structures that are evident on microscopy and that relate to the organisation of actin and myosin protein filaments in muscle cells. Actin and myosin are proteins responsible for muscle contraction. The data suggest that the degree of disruption is dependent on duration and amplitude of sonication. These effects were seen after 1 min of sonication. The minimal temperature rise and the sub-cavitation pressure amplitudes suggest a mechanism other than cavitation and thermal effects for the cell disruption. The authors speculate that the changes may be due to acoustic streaming and movement due to radiation forces. They point out that the relatively constrained structures in muscle cells are not expected to be subjected to these forces. However, in the presence of non-uniform sound fields, twisting and stretching of the membranes and filamentous structures may occur, due to viscous stresses.
In succession of studies on plant cells by M.W. Miller [69
], D.G. Miller subjected Elodea
cells to ultrasound for 100 s at frequencies ranging from 0.45 to 10 MHz [71
]. Intensity thresholds for cell death were found to vary with frequency and ranged from 75 mW cm
at 0.65 MHz to 180 mW cm
at 5 MHz. Cell death was attributed to the presence of gas bodies in inside the Elodea
leaves, modelled in a separate paper [72
]. The ultrasound-induced motion of fluids had been reported earlier inside Elodea
] and in a Curcurbita pepo
hair cell [74
Discoid platelet suspensions were subjected to ultrasound fields in the 1–10-MHz frequency range at acoustic pressure amplitudes in the range of 0.5–76 kPa [75
]. Acoustic streaming was observed and changes in transmitted light intensity were attributed to changing platelet orientation. Platelet disruption had been reported after 5 min of 1-MHz sonication at intensities of 0.2 and 0.6 W cm
]. In the latter study, following sonication, platelet debris were observed and platelet function was impaired. There were qualitative differences between sonicated and non-sonicated specimens in the macroscopic characteristics of the clot.
This concludes the overview of early cellular acoustics studies. Despite the high intensities used, not all phenomena observed are destructive, most notably intracellular streaming.
This section gives an overview of cellular acoustics studies that resulted in transient or permanent cell damage. The studies have been treated in chronological order.
Human red blood cells were put in dialysis tubing and exposed to 1 to 2 min of 1-MHz continuous-wave ultrasound at intensities of 0–5 W cm
spatial peak temporal average [77
]. Some of the samples contained ultrasound contrast agent. The degree of cell lysis was found to be intensity-dependent. The maximum lysis was over 50% with ultrasound contrast agent present and 30% without ultrasound contrast agent at an intensity of 3 W cm
. After this maximum, with increasing intensity, there was some decline in lysis. The degree of lysis was found to be insensitive to the concentration of ultrasound contrast agent but decreased with increasing red blood cell concentrations (haematocrit).
Rendering cell membranes permeable to large molecules such as proteins and deoxyribonucleic acid (DNA) has potential therapeutic applications [78
]. The effects of ultrasound on cell membrane permeation was studied by subjecting bovine red blood cells to continuous ultrasound at 24 kHz. A number of incident pressures were used in this study under various conditions and were estimated to range from less than 1 atm to 10 atm. Permeation was determined by measuring the haemoglobin released, which was found to increase as a linear function of incident pressure. It was also found to increase as a function of sonication time, with a threshold for permeation of 100 ms.
A human leukaemia-60 (HL-60) cell line in suspension was exposed to continuous-wave 255-kHz ultrasound at an intensity of 0.4 W cm
for 30 s [79
]. Experiments were performed with and without the photosensitive drug merocyanine 540. Scanning electron microscopy showed that, in the presence of merocyanine 540, there were substantial disruptions to the cell membrane including porosity, dimpling craters, and breaches. In the absence of the drug, however, minimal membrane changes were observed. In addition, inclusion of merocyanine 540 in the ultrasound experiments resulted in a measurable diminution in cell viability which was not seen in the absence of the drug. This study is the reason that sonic cell permeation is referred to as sonoporation.
Belgrader et al. investigated the effect of ultrasound sonication on the spore-forming bacterium B. subtilis
, which served as an anthrax spore (B. anthracis
) surrogate [80
]. The study aimed at identifying suitable techniques to disrupt anthrax spores, a critical step in achieving rapid and sensitive genetic identification, e.g., polymerase chain reaction (PCR), in cases where B. anthracis
spores are used as a weapons. These spore-forming bacteria have an outer cortex that is extremely resistant to disruption (lysis) by various physical and chemical techniques, and the goal was to identify a technique that could achieve rapid spore lysis. Spore disruption was achieved by incubating the spores with 106-
m glass beads to enhance the destructive effects of cavitation. Sonication using 60 W at 67 kHz for 2 min, 50 W at 22 kHz for 30 s, and 40 W at 47 kHz for 30 s were tested. In their final design, adequate cell lysis was achieved in 30 s.
In a follow-up study [81
], spores of the same bacterium B. subtilis
were subjected to sonication at 40 kHz in the presence of 106-
m glass beads for 10 to 20 s. The acoustic horn amplitudes used were expressed in its surface displacement, 25 and 38
m peak-to-peak, and the sonicated liquid was subjected to hydrostatic pressures ranging from 34 to 138 kPa, in an attempt to improve coupling between the horn and the liquid. The ultrasonic energy was transferred from the horn to the fluid via a thin flexible membrane and it was shown that this design avoids cavitation as large pressure drops were absent due to the film separating from the horn. The sonication technique produced effective disruption of bacterial spores as evidenced by scanning electron microscopy. The utility of this technique as a means of releasing DNA from disrupted spores for the purpose of molecular diagnostics was discussed and it was concluded that the efficiency of DNA amplification using PCR increased as a function of the applied hydrostatic pressure.
In a leukaemic cell study [82
], therapeutic ultrasound at a frequency of 750 kHz and spatial-peak temporal average intensity levels of 103.7 W cm
and 54.6 W cm
was applied to HL-60, K562, U937, and M1/2 leukemia cell line cultures. Lower acoustic amplitudes of 22.4 W cm
spatial-peak temporal-average intensity were used as a control. Although it is well known that high-intensity ultrasound causes inertial cavitation effects, the authors were able to demonstrate the induction of programmed cell death, i.e., apoptosis, which holds promise for cancer therapy. Interestingly, the effects were similar to those produced by gamma radiation.
Cultured vascular endothelial cells were subjected to eight repetitions of sonication for 1 min each experiment with unspecified high-frequency ultrasound at 2.5 W cm
in the presence of plasmid DNA with and without an ultrasound contrast agent present [83
]. In addition, in-vivo sonication under similar conditions was performed for 2 min on a damaged rat carotid artery in the presence of DNA. In both cases, transfection of DNA into cells was achieved with higher efficiency under sonication, and even more so in the presence of the ultrasound contrast agent. This paper is generally considered as fundamental proof that cells themselves may respond to ultrasound at acoustic conditions below the inertial cavitation threshold.
The cytotoxic effect of low-energy ultrasound at 7 mW mL
acoustical power was evaluated for various exposure times ranging from 30 min to 5 h at a frequency of 1.8 MHz and on/off cycle of 5.5 ms/3 ms [84
]. Normal mononuclear cells, primary leukaemic cells and four leukaemic cell lines were studied. The authors demonstrated that necrosis is significantly diminished while apoptosis is stimulated in leukaemic cells. They also demonstrated that ultrasound exposure is linked to oxidative stress, and that active oxygen scavengers reduce the effect of ultrasound on apoptosis, suggesting a sonochemical mechanism.
A bacterium E. coli
and a yeast species S. cerevisiae
were studied under ultrasound sonication with a view to producing cell lysis as a first step in various diagnostic processes [85
]. Sonication was achieved using a spherically focussed 1-MHz ultrasound beam in a specially designed sonication chamber. Treatment was at 5.2 W cm
for 30 s, which resulted in greater than 99% loss in viability of both cell types. However, the yeasts demonstrated a relative resistance to disruption and further chemical techniques were needed to liberate cell contents.
The transfer of DNA into cells has clinical, bio-industrial and environmental applications [86
]. This study investigated the use of 40-kHz ultrasound to achieve DNA transfer into a variety of bacterial species. In the centre of the experimental bath used, the estimated intensity was 240 mW cm
. The optimal duration of sonication was chosen to be 10 s, as an optimal compromise between the competing requirements of efficiency of DNA transfer and minimising ultrasound-induced loss of cell viability. Sonication is the putative mechanism for ultrasound-based DNA transfer and it proved substantially more efficient than the commonly used methods of electroporation and conjugation.
The combined effects of low-intensity pulsed ultrasound and doxorubicin (DOX) on cell killing and apoptosis induction of human myelomonocytic lymphoma U937 cells was investigated in vitro [87
]. The experiments were conducted in four groups, including an ultrasound-treated group and a combined ultrasound and DOX-treated group. Cells were exposed to 5
M DOX for 30 min and sonicated 60 s by 1-MHz pulsed ultrasound with a 100-Hz pulse-repetition frequency and a 10% duty cycle. The acoustic intensities varied between 0.2 and 0.5 W cm
. No cell killing or induction of apoptosis was observed at 0.2 W cm
. However, cell killing, induction of apoptosis, and hydroxyl radical formation were detected at intensities equal to and greater than 0.3 W cm
. More radicals were produced in the combined ultrasound and DOX group than with ultrasound alone. Yoshida et al. hypothesised that DOX treatment weakens cell membranes, so that sonoporation is more successful.
Sonication with 40-kHz ultrasound was shown to inhibit growth of Gram-negative bacteria with species such as E. coli
showing sufficient sensitivity that they were eradicated in as little as 5 min [88
]. Gram-positive bacteria, however, were resistant to sonication. This study shows that inhibition of bacteria by sonication is dependent on a number of factors including species, temperature, and duration of sonication. The results have implications for the management of bacterial infections of prosthetic implants which represent an important cause of morbidity and implant failure.
In a study on the ultrasound-induced inactivation of Gram-negative and Gram-positive bacteria in secondary treated municipal waste water [89
], various bacterial species were subjected to 24 kHz of 1500 W L
, corresponding to 5400 kJ L
specific nominal energy, for a duration of 60 min in the presence and absence of titanium dioxide particles. Gram-negative bacteria proved to be highly susceptible to inactivation by sonication using this regime, showing a response of greater than 99%. Gram-positive bacteria showed substantially lower inactivation rates. Adding titanium dioxide enhanced the response of both Gram-negative and Gram-positive bacteria to the destructive effect of sonication. However, this enhancement was far more modest in the case of Gram-positive bacteria.
To investigate the effect of diagnostic ultrasound on blue-green algae eradication in a laboratory setup [90
], three undamped single-element ultrasound transducers were used, with centre frequencies of 200-kHz, 1-MHz, and a 2.2-MHz, respectively. The transducers were subjected to 16-Vpp square pulses at an 11.8-kHz pulse-repetition rate. Low acoustic amplitudes were used in order to comply with an MI below 0.3. The peak-negative acoustic pressures were 40 kPa for the 1-MHz transducer and 68 kPa for the 2.2-MHz transducer, respectively. The blue-green algae used were of the Anabaena sphaerica
species. Blue-green algae were forced within minutes to sink at the ultrasonic frequencies studied, thus supporting the hypothesis that heterocysts release gaseous nitrogen during sonication. A similar study had been done on a different cyanobacteria species, Microcystis aeruginosa
]. Beakers were sonicated during 5 min at 25 kHz at 0.32 W mL
, which inhibited growth. Fourteen days after sonication, the cell concentration was only 14.1% of the control sample.
To investigate the effect of low-intensity ultrasound on DNA [92
], 1.0-MHz ultrasound with 100-Hz fixed pulse-repetition frequency and 10% duty cycle was generated during 1 min in culture dishes containing four different leukaemia cell lines, U937, Molt-4, Jurkat, and HL-60, at intensities of 0.1, 0.2, 0.3, and 0.4 W cm
, corresponding to acoustic pressure amplitudes of 0.061, 0.105, 0.132, and 0.144 MPa, respectively. Only at the two highest intensities were DNA double-strand breaks with all cell lines observed. This damaging effect was attributed to mechanical stress.
A modified experimental setup was used to carry out ultrasound-assisted gene transfection [93
]. Sonication was carried out using 1.0-MHz ultrasound at an intensity of 0.3 W cm
and a 50% duty cycle with a 5-Hz pulse-repetition frequency. Dishes containing HeLa cells in the presence of free plasmid DNA (pDNA) were sonicated immediately after preparation for periods of 30 s or 15 min. The results showed that ultrasound enhances the intracellular trafficking of previously internalised genes when longer sonication periods are applied.
There is a need to have techniques to disrupt (lyse) cells in order to release their contents for the purpose of drug development and other biological research [94
]. Detergent-based disruption frequently has unfavourable effects on cells. Ly et al. developed an ultrasonic method to affect this disruption. Medical Research Council cell strain 5 (MRC-5) cells infected with an attenuated Varicella–Zoster virus were subjected to ultrasound of intensities from 0.1–10 W cm
and at duty cycles of 0.1–20%. Cell lysis was achieved.
Most experimental studies in this section resulted in lysis. If we regard sonoporation as unsuccessful lysis, we might explain why both phenomena are observed in the same acoustic regimes. Apoptosis is less often observed. This phenomenon is associated with high MI and the formation of free radicals.
The experimental studies with bacteria were performed under unclear conditions. It would be interesting to compare acoustic lysis thresholds for mixtures of desirable and undesirable cells.
This section gives an overview of cellular acoustics studies that resulted in the translation of cells. The studies have been treated in chronological order.
To investigate the effects of ultrasound on blood platelets, platelet-rich plasma and washed platelets were treated with 22-kHz ultrasound at intensities ranging from 1 to 8.8 W cm
over multiple seconds [95
]. Sonication of a calcium-containing preparation resulted in intensity- and time-dependent platelet aggregation. This effect, however, was absent in a calcium-free medium. Both the calcium-containing and calcium-free preparations showed a substantial increase in intracellular calcium in response to sonication.
Zourob et al. addressed the need to reliably and sensitively detect bacteria without the time-consuming step of culture [96
]. A sonication and detection chamber was constructed. The chamber included an ultrasound-producing piezoelectric transducer and a specially designed optical metal-clad leaky waveguide (MCLW) with immobilised antibodies on the surface which, using optical techniques, served as the bacterial detector. This MCLW detector was created by depositing the cladding on a 1-mm glass slide that served as a half-wavelength ultrasound reflector, resulting in ultrasound standing waves, with a node forming at the detector-water interface at a frequency of 3 MHz. This caused bacterial spores to collect at the detector during sonication through radiation forces. Stepping the ultrasound in 20-kHz increments from 2 to 4 MHz, it was found that 2.94 MHz was a suitable operating frequency as it represented the maximum voltage difference between the water-filled and empty chamber and was assumed to represent a resonance in the water. As the chamber was a quarter wavelength long, only a single node could form. Sonication for 3 min caused the bacterial spores to move efficiently towards the detector and form regular patterns at the detector surface. These patterns varied in their appearance with ultrasound frequency changes as small as 150 kHz. Increasing the applied peak-to-peak voltage, which is proportional to sound pressure, resulted in increased bacterial spore capture at the interface up to a maximum of 4 V, after which the bacterial spore capture diminished due to the formation of aggregates.
In a similar experimental setup as the one used by Mizrahi et al. [97
], endothelial cells were subjected to 1-MHz ultrasound with acoustic pressure amplitudes between 50 kPa and 300 kPa and a duration of 5 min at a 20% duty cycle [26
]. Endothelial detachment was observed, followed by the geometric reorganisation of the cells according to a periodic pattern, corresponding to nodes or antinodes of the sound field. In addition, sonication caused increased clustering of
3 integrin, a transmembrane protein. However, sonication did not change the amount of
-actin monomers, which are involved in reshaping of the cell.
The accumulation of cells in the nodes or antinodes of a sound field was also described in a study to separate bacterial E. coli
cells and yeast cells, sonicated at 1 MHz and 3 MHz [98
Following an early review on acoustic manipulation [15
], several studies were published on devices that use sound to force cells to translate. Optical observations of the clustering behaviour of living cells and several other particles were done in a standing sound field at 1 MHz or 3 MHz continuous-wave ultrasound with peak-to-peak amplitudes between 1 V and 10 V, generated inside a ring transducer [99
]. Upon sonication, blood cells were observed to become trapped in the nodes of the ultrasound field owing to primary radiation forces. It was found that red blood cells and hydrophobic particles translate like a particle trapped inside a thin gas shell. In fact, the sonophore model mentioned treats biological cells in a similar way [58
]. Cells have also proven to be responsive to acoustic radiation forces at a frequency much higher than used in the former study [99
], making single cell-type-specific cell sorting feasible [100
]. The latter studies used a device operating with standing waves of 19.4 MHz [100
]. A similar device for the identification, separation and cell-type specific manipulation of not single but multiple biological cells was also designed and built [102
]. Cultured cells in a Petri dish were sonicated at 7-MHz continuous-wave ultrasound for 30 s. After sonication, Chinese hamster ovary (CHO) cells were seen to have formed clusters of packed cells whist human embryonic kidney (HEK) cells did not show cluster formation at all. The experiments were done with separate CHO and HEK cell cultures, and a mixture thereof. In a mixture of both CHO and HEK cell cultures, only the cells of one type cluster. It was concluded that different cell types may behave differently at the same ultrasound frequency.
Most experimental studies resulting in translation were carried out in standing wave fields. Therefore, cell aggregation is the most-observed translation phenomenon. It is interesting to trace cell translation speed as a function of cell bulk modulus. If subtle cell stiffness differences result in a significant speed difference, individual cancer cells or parasite-infested cells might be traced acoustically.
2.5. Internal Changes
This section gives an overview of cellular acoustics studies that resulted in changes inside the cells. The studies have been treated in chronological order.
Rabbit corneas were exposed in vivo to continuous-wave ultrasound at a frequency of 880 kHz and with intensities ranging from 0.19 to 0.56 W cm
for 5 min [106
]. These intensities correspond to ultrasound pressures of 0.08 to 0.13 MPa and mechanical indices of 0.08 to 0.14. The increasing intensities of sonication caused sodium fluorescein, a hydrophilic dye introduced onto the corneal surface, to appear in the fluid of the anterior eye at higher concentrations than in non-sonicated eyes, with concentration increases over non-sonicated eyes ranging from more than double, to more than 10-fold at the highest intensities. Microscopic and macroscopic examination of the corneas after sonication revealed structural changes in the surface layer of cells including pitts, but not in the deeper layers. These changes reversed after approximately 90 min.
Or et al. proposed that relative oscillatory displacements between intracellular structures may explain the effects of low to medium intensity ultrasound on cells and tissues [107
]. Such effects include modulation of action potentials in excitable tissues, modulation of angiogenesis, changes in membrane permeability and modulation of molecular expression. The authors constructed a linear model for a spherical object which is intended to approximate an intracellular structure such as a nucleus embedded within a homogeneous viscoelastic medium. Maximal amplitude vibrations are found in the sub-MHz range with the specific frequencies at which maximum oscillations occur being consistent with resonance phenomena. The authors suggested that the very small intracellular strain associated with these conditions, through a cyclic fatigue-like mechanism may be responsible for the biological effects.
As a first attempt to explain sub-cavitation threshold cellular acoustics [58
], studies in which fish epidermis cells had been subjected to both cavitation-inducing 1-MHz, and non-cavitation inducing 3-MHz continuous-wave ultrasound [108
] were re-examined. It was shown that there is a graded range of biological effects which includes behaviours that do not involve cavitation. The authors were also able to demonstrate ultrasound-induced changes to cellular organelles. The cellular response to ultrasound was attributed to the formation of gas bubbles inside the bilayer cell membrane, the so-called sonophore hypothesis [58
In a follow-up study by the same group [97
], real-time in-vitro microscopic studies were performed on cells subjected to uniform pulsed 1-MHz ultrasound with a 20% duty cycle and intensities of 1 W cm
and 2 W cm
, corresponding to hydrophone-measured acoustic pressure amplitudes of 170 kPa and 290 kPa, respectively. Substantial cytoskeletal changes and remodelling were evident at higher intensities corresponding to remarkably small strain values.
In a very recent study on the effect of low-intensity ultrasound and mesenchymal-epithelial (MET) signaling on cellular motility and morphology [109
], continuous-wave low-intensity ultrasound of 200-kPa pressure amplitude at 960 kHz was applied to cells from a Madin–Darby canine kidney (MDCK) cell line. The putative basis for the effects on the MDCK cell membrane is the bilayer sonophore model whereby intramembrane cavitation occurs at moderate acoustic amplitudes. The authors have demonstrated that their setup results in modulation of the so-called MET tyrosine kinase signalling pathway which in turn modifies cell morphology and diminishes critical cancer cell behaviour such as motility. This may form the basis of novel cancer therapies.
The experimental studies in this section were performed at MI < 0.3. Low-amplitude sonication causes subtle intracellular effects. Follow-up research must validate the sonophore hypothesis or provide an alternative explanation for the phenomena observed.