Dmitry Grishenkov

Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging

Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

Characterization of acoustic properties of PVA-shelled ultrasound contrast agents : linear properties (Part I)

This work examines the linear acoustic behavior of ultrasound contrast agents made of three types of poly (vinyl alcohol) (PVA) shelled microbubbles manufactured at different pH and temperature conditions. Backscattered power, attenuation coefficient and phase velocity of ultrasonic waves propagating through suspensions of PVA contrast agents were measured at temperature values ranging between 24 degrees C and 37 degrees C in a frequency range from 3 MHz to 13 MHz. Enhancement of the backscattered power higher than 20 dB and displaying a weak dependence on temperature was observed. Attenuation and phase velocity, on the other hand, showed higher sensitivity to temperature variations. A modified version of the Church model, which accounts for the dispersion of the dynamic modulus of the PVA shells, was developed to simultaneously fit the attenuation and phase velocity data at 24 degrees C. The frequency dependence of the storage modulus was found to be that of semiflexible polymeric networks. On the other hand, the frequency dependence of the dynamic loss modulus suggests that additional mechanisms, which may be related to the finite dimensions of the shell and/or to its inhomogeneity, may play a significant role in the dissipation of the acoustic energy. For the microbubbles of interest, this model predicts frequency dependent resonance frequency higher than 100 MHz.

CHARACTERIZATION OF ACOUSTIC PROPERTIES OF PVA-SHELLED ULTRASOUND CONTRAST AGENTS: ULTRASOUND-INDUCED FRACTURE (PART II)

Knowledge of the magnitude of the peak negative pressure, P(thr), at which ultrasound contrast agents fracture is relevant for using these microbubbles both as devices for contrast enhancement purposes, as well as carriers of drugs to be delivered locally. In the second part of this communication, the acoustic properties of three types of microbubbles stabilized by poly (vinyl alcohol) (PVA) shells are further investigated. In particular, the dependence of P(thr) on system parameters such as the number of cycles, frequency and exposure is examined. The effects of temperature, blood and, wherever data are available, of the dimension of the microbubbles on P(thr) are also considered. The large shell thickness notwithstanding, the results of this investigation show that at room temperature, PVA contrast agents fracture at negative peak pressure values within the recommended safety limit. Furthermore, P(thr) decreases with increasing temperature, radius of the microbubbles and number of cycles of the incident wave. Fatigue seems to be a physical mechanism playing a dominant role in the fracture process. The effect of blood on P(thr) varies according to condition under which the microbubbles have been synthesized, although stiffening of the shell is observed in most cases. In conclusion, these results suggest that PVA-shelled microbubbles may offer a potentially viable system to be employed for both imaging and therapeutic purposes.

In vitro contrast-enhanced ultrasound measurements of capillary microcirculation: Comparison between polymer- and phospholipid-shelled microbubbles

The focus of contrast-enhanced ultrasound research has developed beyond visualizing the blood pool and its flow to new areas such as perfusion imaging, drug and gene therapy, and targeted imaging. In this work comparison between the application of polymer- and phospholipid-shelled ultrasound contrast agents (UCAs) for characterization of the capillary microcirculation is reported. All experiments are carried out using a microtube as a vessel phantom. The first set of experiments evaluates the optimal concentration level where backscattered signal from microbubbles depends on concentration linearly. For the polymer-shelled UCAs the optimal concentration level is reached at a value of about 2×10(4)MB/ml, whereas for the phospholipid-shelled UCAs the optimal level is found at about 1×10(5)MB/ml. Despite the fact that the polymer shell occupies 30% of the radius of microbubble, compared to 0.2% of the phospholipid-shelled bubble, approximately 5-fold lower concentration of the polymer UCA is needed for investigation compared to phospholipid-shelled analogues. In the second set of experiments, destruction/replenishment method with varied time intervals ranging from 2ms to 3s between destructive and monitoring pulses is employed. The dependence of the peak-to-peak amplitude of backscattered wave versus pulse interval is fitted with an exponential function of the time γ=A(1-exp(-βt)) where A represents capillary volume and the time constant β represents velocity of the flow. Taking into account that backscattered signal is linearly proportional to the microbubble concentration, for both types of the UCAs it is observed that capillary volume is linearly proportional to the concentration of the microbubbles, but the estimation of the flow velocity is not affected by the change of the concentration. Using the single capillary model, for the phospholipid-shelled UCA a delay of about 0.2-0.3s in evaluation of the perfusion characteristics is found while polymer-shelled UCA provide response immediately. The latter at the concentration lower than 3.6×10(5)MB/ml have no statistically significant delay (p<0.01), do not cause any attenuation of the backscattered signal or saturation of the receiving part of the system. In conclusion, these results suggest that the novel polymer-shelled microbubbles have a potential to be used for perfusion evaluation.

Characterization of ultrasound-induced fracture of polymer-shelled ultrasonic contrast agents by correlation analysis

Beyond a characteristic value of the negative peak pressure, ultrasound fracture the shell of ultrasonic contrast agents (UCAs). Existing criteria for ascertaining this threshold value exploit the dependence of the amplitude of the UCA acoustic response on the incident pressure. However, under the common experimental conditions used in this work, these criteria appear to be unreliable when they are applied to UCAs that are stabilized by a thick polymeric shell. An alternative criterion for determining the onset of shell fracture is introduced here, which uses variations of the shape of the acoustic time-domain response of an UCA suspension. Experimental evidence is presented that links the changes of the cross-correlation coefficient between consecutive time-domain signals to the fracture of the shells, and consequent release of air microbubbles. In principle, this criterion may be used to characterize similar properties of other types of particles that cannot undergo inertial cavitation.

Graphene Meets Microbubbles: A Superior Contrast Agent for Photoacoustic Imaging

Coupling graphene with a soft polymer surface offers the possibility to build hybrid constructs with new electrical, optical, and mechanical properties. However, the low reactivity of graphene is a hurdle in the synthesis of such systems which is often bypassed by oxidizing its carbon planar structure. However, the defects introduced with this process jeopardize the properties of graphene. In this paper we present a different approach, applicable to many different polymer surfaces, which uses surfactant assisted ultrasonication to exfoliate, and simultaneously suspend, graphene in water in its intact form. Tethering pristine graphene sheets to the surfaces is accomplished by using suitable reactive functional groups of the surfactant scaffold. We focused on applying this approach to the fabrication of a hybrid system, made of pristine graphene tethered to poly(vinyl alcohol) based microbubbles (PVA MBs), designed for enhancing photoacoustic signals. Photoacoustic imaging (PAI) is a powerful preclinical diagnostic tool which provides real time images at a resolution of 40 μm. The leap toward clinical imaging has so far been hindered by the limited tissues penetration of near-infrared (NIR) pulsed laser radiation. Many academic and industrial research laboratories have met this challenge by designing devices, each with pros and cons, to enhance the photoacoustic (PA) signal. The major advantages of the hybrid graphene/PVA MBs construct, however, are (i) the preservation of graphene properties, (ii) biocompatibility, a consequence of the robust anchoring of pristine graphene to the bioinert surface of the PVA bubble, and (iii) a very good enhancement in a NIR spectral region of the PA signal, which does not overlap with the signals of PA active endogenous molecules such as hemoglobin.

MicroBubble activated acoustic cell sorting

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary, air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes and can be used to selectively migrate target cells. As a proof of principle, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique expands the acoustic cell manipulation possibilities and offers cell-sorting solutions suited for applications at point of care.

Acoustic characterization and contrast imaging of microbubbles encapsulated by polymeric shells coated or filled with magnetic nanoparticles

The combination of superparamagnetic iron oxide nanoparticles with polymeric air-filled microbubbles is used to produce two types of multimodal contrast agents to enhance medical ultrasound and magnetic resonance imaging. The nanoparticles are either covalently linked to the shell or physically entrapped into the shell. In this paper, the characterization of the acoustic properties (backscattered power, fracturing pressure, attenuation and dispersion of the ultrasonic wave) and ultrasound imaging of the two types of magnetic microbubbles are presented. In vitro B-mode images are generated using a medical ultrasound scanner by applying a nonconventional signal processing technique that is suitable to detect polymeric bubbles and based on the combination of multipulse excitation and chirp coding. Even if both types of microbubbles can be considered to be effective ultrasound contrast agents, the different structure of the shell loaded with nanoparticles has a pronounced effect on the echogenicity and the detection sensitivity of the imaging technique. The best results are obtained using microbubbles that are externally coated with nanoparticles. A backscattered power of 20 dB was achieved at lower concentration, and an increment of 8 dB in the contrast-to-tissue ratio was observed with respect to the more rigid microbubbles with particles entrapped into the shell.

Unique pumping-out fracturing mechanism of a polymer-shelled contrast agent: an acoustic characterization and optical visualization

This work describes the fracturing mechanism of air-filled microbubbles (MBs) encapsulated by a cross-linked poly(vinyl alcohol) (PVA) shell. The radial oscillation and fracturing events following the ultrasound exposure were visualized with an ultrahigh-speed camera, and backscattered timedomain signals were acquired with the acoustic setup specific for harmonic detection. No evidence of gas emerging from defects in the shell with the arrival of the first insonation burst was found. In optical recordings, more than one shell defect was noted, and the gas core was drained without any sign of air extrusion when several consecutive bursts of 1 MPa amplitude were applied. In acoustic tests, the backscattered peak-to-peak voltage gradually reached its maximum and exponentially decreased when the PVA-based MB suspension was exposed to approximately 20 consecutive bursts arriving at pulse repetition frequencies of 100 and 500 Hz. Taking into account that the PVA shell is porous and possibly contains large air pockets between the cross-linked PVA chains, the aforementioned acoustic behavior might be attributed to pumping gas from these pockets in combination with gas release from the core through shell defects. We refer to this fracturing mechanism as pumping-out behavior, and this behavior could have potential use for the local delivery of therapeutic gases, such as nitric oxide.

Ultrasound contrast agent loaded with nitric oxide as a theranostic microdevice

The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications (ie, theranostics) of myocardial ischemia. We used gas-filled MBs with an average diameter of 4 μm stabilized by a biocompatible shell of polyvinyl alcohol. In vitro acoustic tests showed sufficient enhancement of the backscattered power (20 dB) acquired from the MBs’ suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1,517 m/s) were comparable with those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared with clinically approved contrast agents with a fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal to 36 minutes. The application of high-power ultrasound pulse (mechanical index =1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 minutes in air-saturated solution and from 17 to 10 minutes in degassed solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6–8 hours. This is a suitable time for initial diagnostics, treatment, and monitoring of the therapeutic effect using a single injection of the proposed multifunctional MBs.