Published: June 12th, 2021
The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.
Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.
Ultrasound is the most widely used medical imaging technique. It is non-invasive, fast, safe, cost-effective, and portable1,2,3. However, blood is a poor ultrasound scatterer, and the contrast of the blood pool can be enhanced by an intravenous injection of ultrasound contrast agents3. This enhanced blood-pool contrast enables the quantification of organ perfusion for diagnostic purposes, e.g., in the detection of coronary artery disease4 and metastatic liver disease5. Indeed, tumor vasculature was proven to be an important prognostic factor6. A major research effort is now directed towards microbubble-assisted, targeted molecular imaging and tailoring contrast agents for therapeutic use.
Commercially available ultrasound contrast agents typically consist of a suspension of coated microbubbles7,8 with diameters ranging from 1 µm to 10 µm9. Since ultrasound contrast agent microbubbles are slightly smaller than red blood cells7, the microbubbles can safely reach even the smallest capillaries without creating an occlusion3. Microbubbles have a dramatically increased ultrasound backscattering coefficient compared to tissue10, owing to their compressible gas core11. Furthermore, the microbubble echo is highly nonlinear, i.e., its spectrum contains harmonics and subharmonics of the driving frequency. In addition, the echo strength is strongly dependent on the resonant response of the bubble12. While tissue scatters only linearly, a small number of microbubbles is sufficient to achieve a high detection sensitivity in harmonic imaging13,14. This nonlinear contrast generation can even be strong enough to track single bubbles in the body15.
The shell of the ultrasound contrast agent stabilizes the bubbles against dissolution and coalescence, thereby increasing their circulation time in the blood pool16. The shell can consist of lipids, polymers, or denatured proteins3,8. It decreases the interfacial tension, thereby limiting the effect of Laplace pressure-driven dissolution17 and creates a resistive barrier against gas diffusion18. To further increase stability, the contrast microbubbles are typically filled with a high-molecular weight gas with low solubility in blood11. The microbubble shell dramatically changes the response of the microbubbles to ultrasound insonation11. Uncoated gas bubbles have a characteristic resonance frequency that is inversely proportional to their size and the addition of a lipid coating increases the resonance frequency with respect to that of an uncoated buble owing to the intrinsic stiffness of the shell3. Furthermore, the shell dissipates energy through dilatational viscosity, which constitutes the dominant source of damping for coated bubbles3. The stabilizing shell has the additional advantage that it can be functionalized, e.g., by binding targeting ligands to the surface of microbubbles. This targeting enables many applications for these bubbles and, in particular, molecular imaging with ultrasound14,19.
Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Microbubbles oscillating in the confinement of a blood vessel can cause microstreaming as well as local normal and shear stresses on the capillary wall3. At high acoustic pressures, large amplitude oscillations may lead to microbubble collapse in a violent process termed inertial cavitation, which, in turn, may lead to rupture or invagination of the blood vessel20. These violent phenomena can induce bioeffects such as sonopermeation21, enhancing the extravasation of therapeutic drugs into the interstitium across the endothelial wall, either paracellularly or transcellularly. It may also improve the penetration of therapeutic agents through the extracellular matrix of stroma-rich tumors21,22 and biofilms23,24, although this mechanism is still poorly understood26.
Ultrasound-mediated drug delivery has shown promising results both preclinically27,28 and in clinical trials22. Moreover, when used with relatively low-frequency ultrasound (~1 MHz), microbubbles have been reported to locally and transiently increase the blood-brain barrier permeability, thereby enabling drugs to enter the brain parenchyma, both in preclinical and clinical studies29,30,31,32,33,34.
There are generally two approaches to ultrasound-mediated drug delivery: the therapeutic material can be co-administered with the bubbles, or it can be attached to or loaded in the bubble shell28,35,36. The second approach has been shown to be more efficient in terms of drug delivery37. Microbubbles can be loaded with drugs or genetic material encapsulated in nanoparticles (liposomes or polymeric nanoconstructs) attached to the shell or incorporated directly in the microbubble shell35,36. Nanoparticle-loaded microbubbles can be activated by (focused) ultrasound to locally release the nanoparticle payload28,33,38,39,40. If such a microbubble is in direct contact with a cell, it has been shown in vitro that the payload can even be deposited onto the cell cytoplasmic membrane in a process called sonoprinting34,35.
The ultrasound parameter space for microbubble insonation is extensive, and the in vivo biological conditions further add complexity. Thus, the combination of focused ultrasound and nanoparticle-loaded microbubbles poses a challenge in the field of targeted therapeutics.
The aim of this work is to provide protocols that can be used to image, in detail, the response of microbubbles as a function of the ultrasound parameters and to study the mechanisms leading to shell rupture and subsequent release of the fluorescently-labeled shell material. This set of protocols is applicable to microbubbles with shells that contain a fluorescent dye. Figure 1 shows a schematic representation of the polymeric-nanoparticle-and-protein-stabilized microbubbles developed at SINTEF (Trondheim, Norway). These bubbles are filled with perfluoropropane gas (C3F8) and the nanoparticles that stabilize the shell contain NR668, which is a lipophilic derivative of Nile Red fluorescent dye38,43. The nanoparticles consist of poly(2-ethyl-butyl cyanoacrylate) (PEBCA) and are PEGylated. Functionalization with polyethylene glycol (PEG) reduces opsonization and phagocytosis by the mononuclear phagocyte system, thereby extending the circulation time14,44. As a result, PEGylation increases the amount of nanoparticles reaching the target site, thereby improving the efficacy of the treatment16. Figure 2 illustrates how the use of four microscopy methods allows researchers to cover all relevant time and length scales. It should be noted that the spatial resolution achievable in optical microscopy is determined by the diffraction limit, which depends on the wavelength of the light and numerical aperture (NA) of the objective and that of the object illumination source45. For the systems at hand, the optical resolution limit is typically 200 nm. Additionally, intravital microscopy can be used to image on the subcellular level46. For the nanoparticle-and-protein-stabilized microbubbles used in this work, the minimum length scale relevant for intravital microscopy is the size of small capillaries (≥10 µm). In vitro high-speed optical imaging (10 million frames per second) and high-speed fluorescence imaging (500,000 frames per second) experiments are described for single microbubbles. High-speed bright-field imaging at nanosecond timescales is suitable to study the time-resolved radial dynamics of the vibrating bubbles. In contrast, high-speed fluorescence microscopy allows for direct visualization of the release of the fluorescently-labeled nanoparticles. Furthermore, the structure of the microbubble shell can be investigated using Z-stack three-dimensional (3D) confocal microscopy, and scanning electron microscopy (the protocol for the latter is not included in the current work). Intravital microscopy consists in using multiphoton microscopy to image tumors growing in dorsal window chambers to provide real-time information on local blood flow and on the fate of fluorescently-labeled nanoparticles in vivo47. The combination of these microscopy methods ultimately provides detailed insight into the behavior of therapeutic microbubble agents in response to ultrasound, both in vitro and in vivo.
NOTE: All experimental procedures were approved by the Norwegian Animal Research Authorities. Details of materials that were used in the protocol can be found in the Table of Materials.
1. Production of microbubbles
NOTE: In this work, the microbubbles of interest are protein-and-nanoparticle-stabilized microbubbles, for which the production protocol has been described previously28,33,48. Therefore, the fabrication protocol has been briefly summarized here.
2. Imaging single bubbles
3. Intravital microscopy
The microbubbles, produced as described in the protocol, were analyzed using various microscopy methods and at various timescales.
The fluorescence of the nanoparticles in confocal microscopy (Figure 6A) indicates that the shell has a non-uniform particle distribution. Other microscopy methods can be used for bubble characterization. For example, Figure 6B shows the overall structure of the microbubble using scanning electron microscopy, as presented in previous work34.
Radial dynamics and phenomenological bubble behavior can be studied using the described in vitro bright-field microscopy method wherein microbubbles were imaged at 10 million frames per second. The radius of single microbubbles was extracted over time using a script written in-house. An example of such a radial response is shown in Figure 7.
An image sequence of typical successful nanoparticle delivery, as described in section 2.3.6, is shown in Figure 8A. The nanoparticles embedded in the microbubble shell can be seen to light up due to fluorescence when the laser light reaches the bubble. Driven by ultrasound insonation, the fluorescent nanoparticles detach from the gas core of the microbubbles and are deposited on the membrane of the sample holder. Finally, the laser is turned off, and the fluorescent nanoparticles are no longer excited. Unsuccessful delivery of the fluorescently-labeled payload of the microbubbles typically looks like the image sequence shown in Figure 8B, where the fluorescent nanoparticles light up on the shell of the microbubble that stays intact during ultrasound exposure.
Real-time intravital multiphoton microscopy during ultrasound was used to investigate the effects of ultrasound and microbubbles on nanoparticle behavior in the blood, enhancement of the permeability of tumor blood vessels, and improvement of the delivery of nanoparticles. The extent and kinetics of penetration into the extracellular matrix as a function of acoustic pressure, frequency, and pulse lengths can be characterized. The effect of the ultrasound treatment may vary with respect to the size and morphology of the vessels and resulting confinement of the bubble. How the ultrasound treatment affects the blood flow and direction can be determined. An example experiment showing the extravasation of nanoparticles over time is shown in Figure 9 at a mechanical index (MI) of 0.826. Results of intravital multiphoton microscopy elucidate the spatial and temporal extravasation of nanoparticles during ultrasound exposure, which is highly beneficial for the complete understanding of the mechanisms underlying ultrasound-mediated delivery of nanoparticles and to optimize such technologies26.
Figure 1: Schematic representation of a microbubble with a shell of fluorescently-labeled polymeric nanoparticles in denatured casein. The microbubbles are typically between 1 µm and 10 µm in diameter. The nanoparticles have a diameter mostly between 100 nm and 200 nm38. Abbreviation: C3F8 = perfluoropropane gas. Please click here to view a larger version of this figure.
Figure 2: Schematic overview showing the relevant time and length scales for bright-field, fluorescence, confocal, and intravital microscopy. Please click here to view a larger version of this figure.
Figure 3: Schematic representation of bright-field microscopy experiments. (A) Experimental setup, (B) the timing diagram, and (C) a typical recorded frame. Scale bar in (C) = 10 µm. Abbreviation: fps = frames per second. Please click here to view a larger version of this figure.
Figure 4: Schematic representation of fluorescence microscopy experiments. (A) Experimental setup, (B) the timing diagram, and (C) a typical recorded frame. Scale bar in (C) = 10 µm. Abbreviation: fps = frames per second. Please click here to view a larger version of this figure.
Figure 5: Schematic representation of intravital microscopy experiments. (A) Experimental setup, (B) the timing diagram, and (C) a typical recorded frame. Scale bar in (C) = 50 µm. Green corresponds to dextran-FITC and red to nanoparticles. Abbreviation: GaAsP = gallium arsenide phosphide. Please click here to view a larger version of this figure.
Figure 6: 3D structure of a single nanoparticle-and-protein-stabilized microbubble. (A) Using confocal microscopy to show the nanoparticles, and (B) using a scanning electron microscope to show the 3D structure. (B) has been reproduced with permission from34. Scale bar in (A) = 5 µm; scale bar in (B) = 2 µm. Please click here to view a larger version of this figure.
Figure 7: Typical spherical oscillations of a 2.89 µm radius nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 1 MHz and an acoustic pressure amplitude of 142 kPa. (A-D) Images from the high-speed recording and the corresponding bubble radius over time curve (bottom). Scale bars = 5 µm, and the red line indicates the initial radius. The illumination profile (arbitrary units) is indicated by yellow. The magnification is 120x. Please click here to view a larger version of this figure.
Figure 8: Image sequence from high-speed fluorescence microscopy. (A) Successful delivery of fluorescently-labeled nanoparticles of a nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 2 MHz and an acoustic pressure amplitude of 600 kPa. (B) Unsuccessful delivery of fluorescently-labeled nanoparticles of a nanoparticle-and-protein-stabilized microbubble insonified at an ultrasound frequency of 2 MHz and an acoustic pressure amplitude of 210 kPa. Scale bars = 10 µm. The magnification is 120x. Please click here to view a larger version of this figure.
Figure 9: Intravital microscopy after insonation of nanoparticle-and-protein-stabilized microbubbles at an ultrasound frequency of 1 MHz and an acoustic pressure amplitude of 800 kPa. (A) Nanoparticles within the vessel, and (B) an image sequence of the area indicated by the white dashed square in (A) depicting the extravasation of dextran (green) and nanoparticles (red). Scale bars = 50 µm. The magnification is 20x. Please click here to view a larger version of this figure.
Different optical microscopy methods were combined to obtain information on the various steps in the delivery of nanoparticles from the surface of microbubbles to the surrounding medium. Imaging of the bubble oscillations was performed, as well as imaging of the release of the nanoparticles from the bubble shell, the extravasation, and the penetration through the extracellular matrix of tumors in vivo. In vitro imaging enables screening of many ultrasound parameters compared to the more complex in vivo setups. The benefit of combining this range of imaging modalities is the complementary information that can be obtained at different timescales - a feature that is crucial to characterize and optimize the microbubbles for successful delivery and to obtain therapeutic efficacy. This approach is useful to understand the delivery mechanisms for all microbubbles alike, including constructs with fluorescently-labeled nanoparticles and drugs.
The most critical steps in the microscopy methods used to study single microbubbles are as follows. For fluorescence microscopy, the nanoparticles should be fluorescently-labeled to enable visualization of the particle release. Furthermore, the sample solution should be diluted enough to isolate single microbubbles for analysis in confocal, bright-field, and fluorescence microscopy methods. In addition, it is important to choose an ultrasound driving frequency and acoustic pressure to excite the bubbles most efficiently, namely at their resonance. If the research question concerns delivery of the nanoparticle payload, the appropriate ultrasound parameters should be part of the investigation. Next to resonance, these bubbles should also be driven at or beyond their threshold for nanoparticle release, typically at relatively high acoustic pressure amplitudes (MI > 0.3)51. For bright-field microscopy imaging, it is critical to choose a high-speed camera with a sufficiently high framerate to minimize motion blur and to avoid aliasing.
Bright-field microscopy is mainly limited by the imaging framerate and intensity of light sources available, as a higher framerate would give a more detailed time-resolved insight into the bubble dynamics, but requires more intense illumination due to shorter exposure times. To study particle release in more detail, the framerate for fluorescence imaging can, in principle, be increased by increasing the intensity of the laser light. However, absorption of the high-intensity laser light by the fluorescently-labeled microbubbles generates heat, even with high quantum yield dyes. This heat can interfere with the experiments at stake, and in extreme cases, induce photo-thermal cavitation52. Thus, in practice, there is a limit to the applied laser fluence. However, intense laser illumination can also be deliberately used to induce particle release from liposomes53. Temperature influences bubble dynamics and ultrasound response, depending on bubble type54. Therefore, if in vitro and intravital methods are to be compared objectively, the in vitro methods discussed in the protocol should be performed at 37 °C. Another limitation of the in vitro methods discussed in the current paper is that the bubbles are not in a free-field environment, as microbubbles will float below the sample holder membrane. Furthermore, there is a selection bias when imaging single microbubbles. However, performing repeated experiments on single bubbles allows for the investigation of the effect of size and removal of the confounding factor-the size distribution. If the bubble response as a function of size can be understood while the concentration is not too high to prevent bubble-bubble interactions, the response of any arbitrary bubble population can be calculated. Finally, both bright-field and fluorescence microscopy methods provide insight into microbubbles convoluted in a two-dimensional (2D) image. If the research question requires more than 2D imaging, the 3D behavior of the bubbles can be resolved by combining the setup described in the protocol with a sideview setup for multiplane imaging55.
An alternative method to study microbubbles is acoustic characterization56. However, measuring the echo of a single microbubble requires locating and isolating a single microbubble within the ultrasound beam56, which poses a challenge typically tackled by the use of a narrow tube or optical or acoustical tweezers57,58. To size bubbles acoustically, the microbubbles can be insonified in the geometrical scattering regime at frequencies much higher than their resonance frequency, which does not induce volumetric microbubble oscillations59. The use of an "acoustical camera" is such a method to image the radial dynamics of single microbubbles in response to ultrasound, wherein a high frequency ultrasound probe is used to determine the radial response of the bubble to a low-frequency driving wave60. The disadvantage of this method is that it can only be used to determine the relative change of the microbubble radius; hence, another method is needed to determine the absolute bubble radius, e.g., through optical imaging61,62. The disadvantage of methods wherein microbubbles are exposed to ultrasound at frequencies higher than their resonance frequency is that at such high frequencies, the penetration depth is decreased59, limiting the usability for in vivo applications. Other forms of microscopy may also be used to study microbubbles such as scanning electron microscopy, atomic force microscopy, and transmission electron microscopy63. The achievable spatio-temporal resolution of these alternative microscopy techniques, however, is generally more limited, and these techniques have the disadvantage that imaging is performed either before or after ultrasound exposure by off-line analysis and typically present a low throughput63. Another alternative is to use a light scattering method, which can be used to study radial dynamics of single microbubbles in real-time, but has a low signal to noise ratio as compared to acoustic scattering methods64.
Real-time intravital microscopy during ultrasound exposure is a powerful method to acquire new insight on the vasculature, behavior of microbubbles, nanoparticles, or other molecules (such as dextran in this case) during ultrasound exposure. A general limitation when performing real-time intravital microscopy is that only a small area of the tissue is imaged, and the penetration depth of the light into tissue is limited. If the imaged vessels contain very few microbubbles and/or nanoparticles within the field of view, little or no information on the nanoparticle behavior and extravasation can be obtained. In addition, because of the limited field of view, a proper alignment between the light and ultrasound paths is crucial. If the ultrasound pressure is high enough to induce bubble destruction, it is also important to choose a pulse repetition frequency that allows fresh bubbles to reperfuse into the field of view between ultrasound pulses. Moreover, as the ultrasound will be reflected from the cover glass in the window chamber and the objective, placing the transducer at an angle is important to reduce reflections as to prevent the formation of standing waves, which distort the calibrated pressure field. Another practical issue is that the setup needs to have sufficient space to mount the ultrasound transducer and waveguide above or below the objective in the microscope setup. The tumors in the dorsal window chamber will have a limited thickness due to the confining chamber and the cover slip; however, if needed, other models could be used. Examples are skinfold tumors, for instance, in the mammary fat pad65 or abdominal intravital imaging of tumors in the various organs66. Such tumors can be grown orthotopically in the appropriate microenvironment, and as such, present a more clinically relevant case.
The methods described in this work enlighten the potential of fluorescently-labeled microbubbles to study the fundamentals of drug delivery applications using bubbles and ultrasound. This combination of microscopy methods provides valuable insight into the microbubble response to ultrasound insonation and its associated acoustic parameter space and presents a clear view of the microbubble and payload behavior over a relevant range of time and length scales.
The authors declare there is no conflict of interest.
|100 MS/s Dual-Channel Arbitrary Waveform Generator model 8026
|Arbitrary waveform generator (programmable)
|Amplifier, used in window chamber setup
|2 MDa dextran
|Arbitrary wave form generator, used in window chamber setup
|Dimmable AC halogen light source
|Antidote to wake animal
|BD Neoflon 24 G
|Becton Dickinson & Company
|Tail vein catheter
|BNC model 575
|Berkely Nucleonics Corporation
|Branson 2510 Ultrasonic Cleaner
|Laboratoires Mabio International
|Cell culture casette (volume 10 mL, membrane area 25 cm2, membrane thickness 175 µm)
|Laser (5 W, excitation wavelength 532 nm)
|DP03014 Digital Phosphor Oscilloscope
|Actavis Group HF
|Anaesthesia of mouse
|Fetal Bovine Serum
|Supplement for cell culture medium
|Used for alignment
|Antidote to wake animal
|Heated animal holder
|A steel holder where the mouse is positioned on its side in a cavity fitting the size of a mouse, with the window chamber lying flat and immobilized with screws on each side. Below the chamber there is a hole in the holder to secure acoustic contact between the transducer and the skin. The holder is heated to a maximum temperature of 37°C, and the temperature is controlled by feedback from a rectal temperature probe in the mouse. The holder is mounted to an XY positioning stage so the animal can be moved independently to image different areas of the window chamber
|Hyper Vision HPV-X2
|National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, University of Wisconsin
|open source image processing program
|In vivo SliceScope
|Filtered, phosphate-buffered saline solution
|Water immersion objective (magnification 60x, working distance 2 mm)
|Anesthesia of mouse
|Accord Healthcare Limited
|Anesthesia of mouse
|MVS 7010 High Intensity Xenon Strobe
|Single-element focused immersion transducer (center frequency 2.25 MHz, focal distance 1", diameter 1")
|Single-element focused immersion transducer (center frequency 2.25 MHz, focal distance 1.88", diameter 1.25")
|Addition to cell culture medium before implantation of tumor in animals
|Roswell Park Memorial Institute 1640
|Cell culture medium
|Addition to cell culture medium before implantation of tumor in animals
|T 25 basic ULTRA-TURRAX
|IKA laboratory technology
|Oscilloscope, used in window chamber setup
|Precision Acoustics Ltd
|Used in window chamber setup
|Used in window chamber setup
|20x water dipping objective
|XY(Z) translation stages
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