Laser-induced cavitation in nanoemulsion with gold nanospheres for blood clot disruption: in vitro results

Abstract

Optically activated cavitation in a nanoemulsion contrast agent is proposed for therapeutic applications. With a 56°C boiling point perfluorohexane core and highly absorptive gold nanospheres at the oil–water interface, cavitation nuclei in the core can be efficiently induced with a laser fluence below medical safety limits (70 mJ/cm² at 1064 nm). This agent is also sensitive to ultrasound (US) exposure and can induce inertial cavitation at a pressure within the medical diagnostic range. Images from a high-speed camera demonstrate bubble formation in these nanoemulsions. The potential of using this contrast agent for blood clot disruption is demonstrated in an in vitro study. The possibility of simultaneous laser and US excitation to reduce the cavitation threshold for therapeutic applications is also discussed.

Inertial cavitation refers to a gaseous bubble rapidly collapsing and releasing thermal and mechanical energy. It plays a key role in therapeutic ultrasound (US), including noninvasive or minimally invasive destruction of kidney stones [1], tumor treatment [2], and drug delivery [3]. However, initiating cavitation usually requires low-frequency US excitations, which limit spatial resolution, and high US negative pressures delivered by high-power systems, thus raising safety concerns. An alternative is to optically induce nuclei using metal nanoparticles [4–7], which can reduce the threshold for US-based therapies and improve spatial resolution with locally activated cavitation. Reducing the cavitation threshold with laser excitation was discussed in [6], where low-frequency (~1 MHz) US stimulation was combined with laser illumination of plasmonic nanoparticles to activate cavitation. However, since cavitation occurs by overheating the medium surrounding the nanoparticles in aqueous solution, damage to surrounding tissue may still arise.

A recently reported liquid perfluorocarbon nanodroplet containing gold nanorods with a bovine serum albumin shell can be optically triggered to form a bubble [8]. However, the stiff shell limits its response to US exposure, and only short-lived (~μs heating time) bubbles, not inertial nuclei, are created by laser pulses, limiting this approach for therapeutic applications.

In this study, a nanoscale emulsion bead encapsulated with absorptive gold nanoparticles [9] is investigated as a possible agent for laser and US-based therapies at low cavitation thresholds. The nanoemulsion consists of a perfluorohexane core with a boiling temperature of 56 °C and a layer of gold nanospheres (GNSs) at the oil–water interface that can be optically activated to generate cavitation nuclei with low laser fluence. By clustering GNSs at high density on the emulsion bead surface, the optical absorption spectrum is broadened and redshifted, thereby enabling its use in deep tissue at near-infrared wavelengths (700–1100 nm). Compared to plasmonic nanoparticles in aqueous solution, this nanoemulsion bead (NEB) with GNS has a much lower boiling point and requires less energy for vaporization (20 J/g to heat perfluorohexane from 37°C to the boiling point versus 263 J/g for water, and 102 J/g enthalpy of vaporization for perfluorohexane compared to 2260 J/g for water), enabling larger bubble growth for the same absorbed energy. Thus, NEB-GNS can create cavitation events, but potentially with lower risk of heat damage to surrounding tissue.

Furthermore, because NEB-GNS does not have a stiff shell, it is more sensitive to US excitation. As we will show later, a negative pressure within the standard imaging range is enough to create long-lived cavitation in NEB-GNS with no laser illumination. In this respect, the nanoemulsion is similar to conventional US contrast agents, preserving the possibility of US-based cavitation therapy, with the cavitation threshold further reduced by laser illumination.

We have previously demonstrated that NEB-GNS exhibits strongly nonlinear photoacoustic (PA) signal response versus applied laser fluence due to bubble formation with dramatically enhanced thermal expansion [10]. The ultimate goal of the work described here is to confine both laser and US exposures to the medical diagnostic range for deep therapeutic applications (e.g., blood clot disruption) using a portable laser system.

Optically induced bubble formation of this nanoagent is verified using a microscope coupled with a high-speed camera (microphotography), and a potential therapeutic application, blood clot disruption, is also demonstrated in an in vitro study. We also explore and discuss the possibility of manipulating the cavitation threshold for this agent under simultaneous pulsed laser and US excitation.

A wavelength of 1064 nm was chosen since it provides good penetration into the body for most biomedical applications. In addition, inexpensive, compact, high-powered commercial fiber or diode-pumped lasers are available at 1064 nm that can operate at kilohertz repetition rates, potentially easing clinical translation of diagnostic and therapeutic applications. As shown in [9], the spectrum of NEB-GNS exhibits significant absorption at 1064 nm.

Colloidal GNSs (about 12 nm in diameter) in an aqueous buffer were functionalized with a dosage of 5.0 polyethylene glycol (PEG)-thiol chains/nm² Au and 500 butanethiol (Sigma Aldrich, St. Louis, Missouri) molecules/nm² Au [11]. NEB-GNSs were produced by adding a dispersion of gold clusters into deionized water with perfluorohexane (Sigma Aldrich, St. Louis, Missouri), and sonicated 1 s on, 4 s off, for a total of 1 min [9,11]. The mean size of a single emulsion bead measured by dynamic light scattering was about 150 nm in diameter.

To view bubble formation resulting from a single laser pulse acting on NEB-GNS, an inverted microscope (TE2000-U, Nikon, Japan) with a 40× water immersed objective (MRD07420 CFI Apo 40X W NIR, Nikon, Japan) was coupled to a high-speed camera (Imacon 200, Hadland Imaging LLC, Santa Cruz, California) and synchronized with a semiconductor fiber laser (HM 40 W G3.1, SPI, South Hampton, UK) delivering a 9 ns, 1064 nm laser pulse, resulting in a fluence of about 70 mJ/cm². NEB-GNS with a concentration of 7.6 × 10¹⁰ beads/ml was sandwiched between plastic food wrap membranes with a thickness of about 300 μm and co-aligned with the objective in a water tank. The high-speed camera can record bubble generation and dynamics with a frame rate up to 2 × 10⁸ frames/s.

Figure 1 shows a series of images of bubble formation induced by a single laser pulse delivered at 120 ns relative to the start of recording. Before laser firing, small dark spots, corresponding to aggregated NEB-GNS or large coalesced beads, were observed (0–100 ns). Note that the microscope resolution (~1 μm) is only sufficient to see aggregated or coalesced NEB-GNS but not a single original bead. The image at the 200–300 ns window clearly shows generated microbubbles. Some expanded to a maximum diameter of about 10 μm in the image at 600–700 ns and then shrank. The bubble size changes with the number of beads in a cluster or the size of a coalesced bead. All bubbles were lost after 1400 ns (~1300 ns after the laser pulse), indicating that their lifetime is about 1 μs. They may disappear due to collapse (inertial cavitation) or recondensation back into the liquid state.

Fig. 1.

Images by a high-speed camera, showing micrometer-sized bubble formation from NEB-GNS with one short (9 ns) laser pulse irradiation at 120 ns. The lifetime of the bubbles is about 1 μs.

As a first application, the potential of using NEB-GNS for human blood clot disruption was investigated in an in vitro experiment. Fibrin gels were prepared according to the procedure described previously [12]. Briefly, gels were formed by mixing CaCl₂, human α-thrombin, and fibrinogen (both from Enzyme Research Laboratories, South Bend, Indiana), with a final concentration of 4 mg/ml fibrinogen. They were injected into a 2.5 mm tube (SLTT-10, Zeus, Orangeburg, South Carolina) and allowed to gelatinize for at least 1 h before performing the experiment.

A small amount (~30 μl) of NEB-GNS with a concentration of about 1.17 × 10¹² beads/ml (0.015 vol. % Au, 1 vol. % perfluorohexane) was added next to a 15-mm-long fibrin clot in the tube. The tube was then connected to a water head on the NEB-GNS side, providing a pressure at the lower end of that in a human artery, 80 mmHg, and immersed in a water tank for US pulse-echo and PA monitoring.

As an initial demonstration of clot disruption with laser-activated cavitation from NEB-GNS, an optical parametric oscillator (OPO) laser (Surelite OPO plus, Continuum, Santa Clara, California) with a wavelength tuned to 750 nm, closer to the peak absorption wavelength of NEB-GNS, was used. Laser pulses (7 ns at 1/e level, 20 Hz pulse repetition rate) were coupled into a fiber bundle (77526, Oriel Instruments, Stratford, Connecticut) to irradiate the emulsion–clot interface, resulting in a 3 mm diameter beam and a fluence of about 7 mJ/cm². Both the fluence and average irradiance are well within guidelines for safe operation at this wavelength [13]. A linear US array transducer (AT8L12-5 50 mm, Broadsound, Hsinchu, Taiwan) interfaced with a Verasonics imaging system (Redmond, Washington) received PA signals and also performed US pulse-echo imaging. A digital camera (DSC W230, Sony, Japan) also recorded the process.

An integrated video including US, PA, and camera movies showing clot disruption is presented in Fig. 2 (Media 1). NEB-GNS (left) and the fibrin clot (right) can be readily seen inside the tube in the camera images. Top and bottom boundaries of the tube can also be observed in the US images but not NEB-GNSs since they are too small to create detectable backscattered signals. Before the laser was turned on, water pressure alone was not strong enough to disrupt the clot.

Fig. 2.

(Media 1) Video of laser-induced fibrin clot disruption with NEB-GNS in a tube using a 750 nm laser with a pulse repetition rate of 20 Hz and a laser fluence of 7 mJ/cm². Two insets show corresponding PA images (left) and US pulse-echo images (right), respectively. The video was played eight times faster than real time.

In US images, bubbles appeared underneath the top tube and blocked the transmission of US to the bottom after the laser started (0:00). In PA images, the interface between NEB-GNS and the clot can be found around −0.5 mm laterally, and heterogeneity due to induced transient bubbles creates high-frequency small PA sources at the left side of the interface (i.e., within NEB-GNS dispersion). Cavitational energy release ruptures the clot surface and breaks fibrin structures, resulting in small pores that help NEB-GNS pushed by water pressure gradually penetrate into the clot (to the right), as shown in the camera movies from 0:00 to 4:24.

When NEB-GNS penetrated into the clot, the interface moved in the PA images from −0.5 to +0.5 mm (~1:40). However, since the laser beam stayed at the interface, PA images could not follow further penetration. For US images, the structure near the top tube boundary changed as NEB-GNS penetrated into the clot, but the bottom boundary remains almost unchanged, indicating a channel created inside the clot instead of pushing the bulk clot to the right. At 4:25, NEB-GNS broke through the clot and flowed to the right, immediately leaving the imaging region. The laser was turned off at 4:45. Both PA and US images were recorded in an interleaved order at a frame rate of 4.6 Hz, and the video was played eight times faster than real time.

Figure 3 (Media 2) shows a camera video (played two times faster than real time) of a clot disruption experiment in a 1.6 mm tube (SLTT-16, Zeus, Orangeburg, South Carolina), repeated with the same 1064 nm fiber laser used for Fig. 1. All the conditions are the same as in Fig. 2, except the laser wavelength changed from 750 to 1064 nm with a high repetition rate of 1 kHz and a pulse width of 250 ns. This configuration gives a pulse energy of 1.33 mJ, which is maximal for this laser. The laser beam is focused to about 0.5 mm in diameter, as indicated with a green dotted circle. In this case, sufficient cavitation microbubbles are formed at a fluence of about 500 mJ/cm² to effectively break the clot. This is a preliminary result aimed to test the feasibility of clot breakage with a portable fiber laser, and it takes less than 40 s to break through the clot. However, both the fluence (500 mJ/cm²) and average radiance are well above safety limits [13]. In addition, depth-dependent light attenuation in turbid biological tissue makes it more difficult to deliver sufficient energy to the target region at centimeter depths. Therefore, there is need for a method that can significantly reduce both the laser fluence and laser-activated cavitation threshold for clot breakage.

Fig. 3.

(Media 2) Camera video of laser-induced fibrin clot disruption with NEB-GNS in a tube using a 1064 nm laser with a pulse width of 250 ns, a pulse repetition rate of 1 kHz, and a laser fluence of 500 mJ/cm². The video was played two times faster than real time.

Recent studies have shown that significantly lower cavitation threshold can be achieved when a laser pulse is delivered to an absorbing medium at the peak-negative pressure of a simultaneous US pulse compared to using either laser or US excitation alone [4–6]. To explore the possibility of manipulating the cavitation threshold of NEB-GNS with combined laser and US excitation, a low-frequency (center frequency at 1.24 MHz) focused transducer (H-102, Sonic Concepts, Bothell, Washington) was used in addition to laser excitation.

First, we explored if NEB-GNS can create inertial cavitation with only US excitation, similar to conventional US contrast agents. The result shows that 10 cycles of US excitation with a 1.5 MPa negative pressure [mechanical index (MI) of 1.35] can generate cavitation nuclei, similar to the cavitational behavior of a typical US micro-bubble agent such as Definity [14]. The probabilities (integrated energy of “slow-time” differential signals of 200 firings compared to the noise threshold) was calculated as 95%, thus demonstrating high sensitivity of NEB-GNS to US excitation.

An experiment using simultaneous laser–US excitation was then performed. The 1064 nm laser (100 Hz repetition rate, 250 ns pulse width, and a significantly reduced fluence, 84 mJ/cm², compared to Fig. 3) was utilized. NEB-GNS with a concentration of 1.26 × 10¹² beads/ml (0.005 vol. % Au and 0.27 vol. % perfluorohexane) were tested in a 1.6 mm tube, which was positioned in the focus of the low-frequency transducer. A homemade un-focused wideband polyvinylidene fluoride (PVDF) transducer (100 kHz–30 MHz bandwidth by 1/e level, 6 mm aperture) detects A-line acoustic signals with and without laser irradiation.

Figure 4 shows preliminary results of simultaneous laser–US excitation of NEB-GNS. Both the original recorded signal and its differential signal are displayed. The peak negative acoustic pressure was 0.97 MPa (MI of 0.87). Note that although the laser fluence of 84 mJ/cm² was higher than 70 mJ/cm² used in Fig. 1, the peak power was too low (more than 20 times smaller due to 250 ns pulse width versus 9 ns) to induce long-lived cavitation bubbles by the laser alone.

Fig. 4.

Preliminary results on manipulating bubble generation by NEB-GNS with simultaneous laser–US excitation. Typical backscattered RF A-line signals and slow-time differential signals received by a broadband transducer when (a), (d) the laser was off, (b), (e) the laser pulse irradiates NEB-GNS at peak positive pressure, and (c), (f) laser pulse was at peak negative pressure.

Figures 4(a) and 4(d) show a sample RF A-line and the corresponding differential signal, respectively, for the case without laser irradiation. The received waveform represents simply the acoustic wave from the low-frequency transducer scattered by the tube with NEB-GNS. The differential signal shows that the residue was at the background noise level. Figures 4(b) and 4(e) show original and differential waveforms, respectively, for the case where the laser pulse irradiated the tube at the arrival time of peak positive pressure. Clearly, the waveform is very close to the previous case, indicating no phase transition in NEB-GNS.

Significant cavitation was observed when laser pulses irradiated the sample at the time of peak negative pressure [Figs. 4(c) and 4(f)]. Before laser incidence, the waveform was unchanged from burst to burst, and the differential signal was at the background noise level. In contrast, the waveform was greatly changed after the laser firing, demonstrating initial cavitation and subsequent bubble expansion and collapse from one laser pulse to the next as a widely varying differential signal. With simultaneous US delivery, the lifetime of the cavitation bubble extended to more than 5 μs (and can be further extended to several milliseconds with longer US excitation), compared to 1 μs for laser excitation alone as shown in Fig. 1. A longer-lived cavitation bubble can be very effective for US-based treatment.

To demonstrate that these snapshots are typical, the cavitation probabilities were obtained from 200 firings as 0%, 3.1%, and 79% for US only, laser at positive peak, and laser at negative peak, respectively, indicating greatly enhanced efficiency of creating inertial cavitation with the laser pulse delivered at peak negative pressure.

In summary, cavitation bubbles were optically induced in NEB-GNS with a single laser pulse, and clot breakage was demonstrated in vitro with the aid of laser-induced bubbles from this agent. Cavitation bubbles were also observed with only ultrasound exposure at 1.5 MPa negative pressure. This feature is unique compared to the hard-shelled nanodroplets described previously [8]. With simultaneous low-frequency US and laser excitation (delivered at peak negative pressure), the cavitation threshold can be reduced and longer-lived bubbles produced, compared to the short-lived ones created with laser excitation alone. This makes NEB-GNS potentially effective for noninvasive therapeutic applications inside the body where the energies of both laser and US excitation are well below safety limits. Next steps include quantifying stable or inertial cavitation activities and determining the cavitation threshold for this new contrast agent by performing parameter sweeps on laser fluence, peak negative acoustic pressure, agent concentration, and so on. We will also explore integrating high-intensity focused ultrasound treatment (i.e., histotripsy) with this nanosystem to further break up and dissolve a clot [15], minimizing potential circulation of small fragments detached from the clot after disruption (i.e., embolism) [16].