Intravascular ultrasound detection and delivery of molecularly targeted microbubbles for gene delivery

We are investigating the combination of microbubble-based targeted drug delivery and intravascular ultrasound (IVUS) imaging as a potential therapy to reduce incidence of restenosis following stent placement in atherosclerotic coronary arteries. The goal of these studies was to determine whether IVUS could be used to detect targeted microbubbles and enhance drug/gene delivery through targeting. Quiescent vascular smooth muscle cells (SMCs) were stimulated with cytokine IL-1β to induce the inflammatory cell surface marker vascular cell adhesion molecule 1 (VCAM-1). Molecular-targeted (VCAM-1 Ab or IgG control Ab), fluorescent-labeled microbubbles were conjugated with plasmid DNA expressing green fluorescent protein (GFP, pMax-GFP) and exposed to the inflamed SMCs under flow to measure adhesion compared with control microbubbles. Gene delivery was performed using a modified IVUS catheter to generate 1.5-MHz ultrasound at 200 kPa. Detection of adherent microbubbles to inflamed SMCs in culture and flow chambers was measured using an IVUS catheter and scanner. VCAM-1-targeted microbubbles enhanced adhesion to inflamed SMCs 100-fold over nontargeted microbubbles. Compared with noninflamed SMCs, VCAM-1-targeted microbubbles exhibited a 7.9-fold increase in adhesion to IL-1β-treated cells. Targeted microbubbles resulted in a 5.5-fold increase in plasmid DNA transfection over nontargeted microbubbles in conjunction with a focused 2.54-cm (1-in) diameter 1-MHz transducer and also enhanced transfection by the modified IVUS transducer at 1.5 MHz. Targeted microbubbles (at a density of 3 × 10? microbubbles/mm2) increased IVUS image intensity 13.2 dB over non-microbubble-coated surfaces. Rupture of microbubbles from the modified IVUS transducer resulted in a 53% reduction in image intensity. Taken together, these results indicate that IVUS may be used to detect targeted microbubbles to inflamed vasculature and subsequently deliver a gene/drug locally.     

Radial modulation of microbubbles for ultrasound contrast imaging

Over the past few years, extensive research has been carried out in the field of ultrasound contrast imaging. In addition to the development of new types of ultrasound contrast agents, various imaging methods dedicated to contrast agents have been introduced, and some of them are now commercially available. In this study, we present results of an imaging technique that is capable of detecting echoes from microbubbles and eliminating those emanating from nonoscillating structures (tissue), thereby enhancing contrast imaging. The method is based on mixing a low frequency (LF) modulator signal and a high frequency (HF) imaging signal to effectively modulate the size of the contrast microbubble through its volumetric oscillations using the LF signal and to probe the radial motion using the HF imaging signal. To evaluate the performances and limitations of the method, high-speed optical observations and acoustic measurements were carried out on soft-shelled microbubbles. The results showed that, by incorporating the modulator signal, the bubbles respond differently compared to the HF excitation alone. The decorrelation between the signals obtained at the compression and expansion phase of the modulator signal is significantly high to be used as a parameter to detect contrast microbubbles and discriminate them from tissue. The echo received from a solid reflector shows identical responses during the compression and rarefaction phase of the LF signal. In conclusion, these results demonstrate the feasibility of this fully linear approach for improving the contrast detection.     

Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification

Recent research has shown that targeted ultrasound contrast microbubbles achieve specific adhesion to regions of intravascular pathology, but not in areas of high flow. It has been suggested that acoustic radiation can be used to force free-stream microbubbles toward the target, but this has not been verified for actual targeted contrast agents. We present evidence that acoustic radiation indeed increases the specific targeted accumulation of microbubbles. Lipid microbubbles bearing an antibody as a targeting ligand were infused through a microcapillary flow chamber coated with P-selectin as the target protein. A 2.0 MHz ultrasonic pulse was applied perpendicular to the flow direction. Microbubble accumulation was observed on the flow chamber surface opposite the transducer. An acoustic pressure of 122 kPa enhanced microbubble adhesion up to 60-fold in a microbubble concentration range of 0.25 x 10(6) to 75 x 106) ml(-1). Acoustic pressure mediated the greatest adhesion enhancement at concentrations within the clinical dosing range. Acoustic pressure enhanced targeting nearly 80-fold at a wall shear rate of 1244 s(-1), suggesting that this mechanism is appropriate for achieving targeted microbubble delivery in high-flow vessels. Microbubble adhesion increased with the square of acoustic pressure between 25 and 122 kPa, and decreased substantially at higher pressures.     

Multiple emulsion microbubbles for ultrasound imaging

In order to improve the sensitivity of ultrasound imaging, the contrast agents, a powerful non-invasive and real-time medical imaging technique, are used. However, air or N₂ or perfluorocarbon only encapsulated microbubbles which are currently used have lower efficiency and short imaging time. So the novel contrast agents with a higher efficiency are required. To achieve this objective, the strategy that we have explored involves the use of superparamagnetic iron oxide (SPIO) Fe₃O₄ nanoparticles multilayer emulsion microbubbles. This multilayer structure consists of three layers. The core is poly-d, l-lactide (PLA) encapsulated N₂ nanobubble with the SPIO nanoparticles forming oil-in-water (W/O) layer. The outermost is water-in-oil-in-water ((W/O)/W) emulsion layer with PVA solution. Herein we describe the synthesis and characterization of ultrasound imaging microstructure with an overall diameter of around 2μm-8μm. On the one hand, the stable gas encapsulated microstructure can provide a high scattering intensity resulting in high echogenicity, On the other hand, SPIO nanoparticles have shown the potential of high-resolution sonography. So the multiple emulsion microbubbles with SPIO can have double action to enhance the ultrasound imaging. Besides, because SPIO can also serve as magnetic resonance imaging (MRI) contrast agents, such microstructure may be useful for multimodality imaging studies in ultrasound imaging and MRI.     

A nanocomplex system as targeted contrast agent delivery vehicle for magnetic resonance imaging dynamic contrast enhancement study

We have developed and tested a liposomal nanocomplex system, which contains Gd-DTPA as a payload and transferrin on the surface, as a tumor specific targeting MRI contrast agent for studying prostate cancer tumors in mice. In vivo, the probe significantly enhanced the MRI signal. The image contrast between the peripheral region of the tumor and the non-involved muscle was nearly 50% higher two hours after administration of the nanocomplex. The liposomal nanocomplex increased the amount of Gd accumulated in tumors by factor 2.8 compared to that accumulated by using Magnevist alone. Moreover, the heterogeneous MRI image features correlate well with the tumor pathology. The image enhancement patterns can be used for cancer prognosis and non-invasive monitoring of the response to therapy.     

Piezoelectric contrast materials for ultrasound imaging

Piezoelectric ceramics and polymers can be used as a type of marker and contrast material for medical ultrasound imaging systems. High-frequency electrical signals are detected from surface electrodes when these materials are introduced into conducting media such as tissue and scanned by ultrasound imaging systems. Detected signals are applied to the imaging circuits of a modified ultrasound system such that they display a unique type of electrical image that shows the piezomaterial's polarization, shape, and position at arbitrarily high contrast compared to the conventional ultrasound acoustic image. The resulting piezoelectric image can be merged in real-time with conventional ultrasound acoustic imaging to form a composite image. This approach is of interest in the development of improved techniques for imaging medical devices that are implanted or otherwise introduced into the body.     

Cargo-towing fuel-free magnetic nanoswimmers for targeted drug delivery

Fuel-free nanomotors are essential for future in-vivo biomedical transport and drug-delivery applications. Herein, the first example of directed delivery of drug-loaded magnetic polymeric particles using magnetically driven flexible nanoswimmers is described. It is demonstrated that flexible magnetic nickel-silver nanoswimmers (5-6 μm in length and 200 nm in diameter) are able to transport micrometer particles at high speeds of more than 10 μm s(-1) (more than 0.2 body lengths per revolution in dimensionless speed). The fundamental mechanism of the cargo-towing ability of these magnetic (fuel-free) nanowire motors is modelled, and the hydrodynamic features of these cargo-loaded motors discussed. The effect of the cargo size on swimming performance is evaluated experimentally and compared to a theoretical model, emphasizing the interplay between hydrodynamic drag forces and boundary actuation. The latter leads to an unusual increase of the propulsion speed at an intermediate particle size. Potential applications of these cargo-towing nanoswimmers are demonstrated by using the directed delivery of drug-loaded microparticles to HeLa cancer cells in biological media. Transport of the drug carriers through a microchannel from the pick-up zone to the release microwell is further illustrated. It is expected that magnetically driven nanoswimmers will provide a new approach for the rapid delivery of target-specific drug carriers to predetermined destinations.     

Simulations and measurements of optical images of insonified ultrasound contrast microbubbles

Ultrasound contrast agents (UCAs) are used in a clinical setting to enhance the backscattered signal from the blood pool to estimate perfusion and blood flow. The UCAs consist of encapsulated microbubbles, measuring 1-10 /spl mu/m in diameter. Acoustic characterization of UCAs is generally carried out from an ensemble of bubbles. The measured signal is a complicated summation of all signals from the individual microbubbles. Hence, characterization of a single bubble from acoustic measurements is complex. In this study, 583 optical observations of freely flowing, oscillating, individual microbubbles from an experimental UCA were analyzed. The excursions during ultrasound exposure were observed through a microscope. Images were recorded with a high frame rate camera operating at 3 MHz. Microbubbles on these images were measured offline, and maximal excursions were determined. A technique is described to determine the diameters of the bubbles observed. We compared the maximal excursions of microbubbles of the same initial size in an ultrasound field with a 500 kHz center frequency at acoustic amplitudes ranging from 0.06 MPa to 0.85 MPa. It was concluded that maximal excursions of identical bubbles can differ by 150% at low acoustic pressures (mechanical index or MI<0.2). At a high acoustic pressure (MI=1.2) an image sequence was recorded on which a bubble collapsed, but an apparently identical bubble survived.     

Bioorthogonal supramolecular cell-conjugation for targeted hitchhiking drug delivery

Cells possess inherent advantages to facilitate targeted payload delivery. Current strategies to conjugate payload carriers to the surface of cells are either via covalent bonds that not only involve complicated synthetic process but also often impair cellular functions, or via biological ligand-receptor interactions that are only specific to particular types of cells. Herein, we report a facile, bioorthogonal supramolecular conjugation strategy to prepare targeted cell-hitchhiking delivery systems, mediated via artificial host–guest interactions between β-cyclodextrin and adamantane, respectively anchored (via insertion) on the surfaces of live cells and payload carriers. In a paw swelling inflammation mouse model, supramolecularly conjugated macrophage-carriers (either cell–cell or cell-nanoparticle systems) were efficiently delivered hand-in-hand to the swelling paw, driven by the inflammatory tropism of macrophage. Furthermore, in an acute lung inflammation model of mouse, supramolecular conjugation of peritoneal macrophage and quercetin-loaded liposomes significantly improved targeting efficiency of the liposomes, and effectively alleviated the lung inflammation through the anti-inflammatory and anti-oxidative effects of quercetin. The cell-friendly, facile, host–guest interactions mediated cellular conjugation may provide the very first general strategy for preparing various cell-hitchhiking delivery systems to meet the needs of diverse biomedical applications.     

Asymmetric oscillation of adherent targeted ultrasound contrast agents

With a lipid shell containing biotin, micron-sized bubbles bound to avidin on a porous and flexible cellulose boundary were insonified by ultrasound. The oscillation of these targeted microbubbles was observed by high-speed photography and compared to the oscillation of free-floating microbubbles. Adherent microbubbles were observed to oscillate asymmetrically in the plane normal to the boundary, and nearly symmetrically in the plane parallel to the boundary, with a significantly smaller maximum expansion in each dimension for bound than free bubbles. With sufficient transmitted pressure, a jet was produced traveling toward the boundary.     

Pharmacytes: an ideal vehicle for targeted drug delivery

An ideal nanotechnology-based drug delivery system is a pharmacyte--a self-powered, computer-controlled medical nanorobot system capable of digitally precise transport, timing, and targeted delivery of pharmaceutical agents to specific cellular and intracellular destinations within the human body. Pharmacytes may be constructed using future molecular manufacturing technologies such as diamond mechanosynthesis which are currently being investigated theoretically using quantum ab initio and density-functional computational methods. Pharmacytes will have many applications in nanomedicine such as initiation of apoptosis in cancer cells and direct control of cell signaling processes.     

Simulation of noninvasive blood pressure estimation using ultrasound contrast agent microbubbles

The microbubble ultrasound contrast agent (UCA) has been widely recognized as a potential noninvasive tool for blood pressure measurement. However, UCA indices such as the shift in the resonance frequency and echo amplitude have problems of low resolution, nonlinear relationship with blood pressure, etc. In this paper, a novel UCA index, the shift in the subharmonic optimal driving frequency (SSODF) of microbubbles, is proposed. The effectiveness of the index for estimating blood pressure was evaluated by performing a microbubble acoustic response simulation. The behavior of commercial UCA microbubbles was investigated as a function of the driving acoustic pressure (in kilopascals) and ambient overpressure (in millimeters of mercury). Simulation results showed that for a 1.6-μm-diameter microbubble, SSODF increased linearly with the overpressure in a range of 0 to 200 mmHg and was maximum (2.07 MHz) at 380 kPa. Changes of the overpressure as small as 5 mmHg can be detected using SSODF. For a population of microbubbles with a Gaussian size distribution (mean diameter: 1.6 μm, standard deviation: 0.2 μm), SSODF was 1.7 MHz at 280 kPa. With further experimental validation, the proposed method may be developed as a novel noninvasive technique for accurate blood pressure measurement.     

Harmonic chirp imaging method for ultrasound contrast agent

Coded excitation is currently used in medical ultrasound to increase signal-to-noise ratio (SNR) and penetration depth. We propose a chirp excitation method for contrast agents using the second harmonic component of the response. This method is based on a compression filter that selectively compresses and extracts the second harmonic component from the received echo signal. Simulations have shown a clear increase in response for chirp excitation over pulse excitation with the same peak amplitude. This was confirmed by two-dimensional (2-D) optical observations of bubble response with a fast framing camera. To evaluate the harmonic compression method, we applied it to simulated bubble echoes, to measured propagation harmonics, and to B-mode scans of a flow phantom and compared it to regular pulse excitation imaging. An increase of approximately 10 dB in SNR was found for chirp excitation. The compression method was found to perform well in terms of resolution. Axial resolution was in all cases within 10% of the axial resolution from pulse excitation. Range side-lobe levels were 30 dB below the main lobe for the simulated bubble echoes and measured propagation harmonics. However, side-lobes were visible in the B-mode contrast images.     

Nano-graphene oxide for cellular imaging and drug delivery

Two-dimensional graphene offers interesting electronic, thermal, and mechanical properties that are currently being explored for advanced electronics, membranes, and composites. Here we synthesize and explore the biological applications of nano-graphene oxide (NGO), i.e., single-layer graphene oxide sheets down to a few nanometers in lateral width. We develop functionalization chemistry in order to impart solubility and compatibility of NGO in biological environments. We obtain size separated pegylated NGO sheets that are soluble in buffers and serum without agglomeration. The NGO sheets are found to be photoluminescent in the visible and infrared regions. The intrinsic photoluminescence (PL) of NGO is used for live cell imaging in the near-infrared (NIR) with little background. We found that simple physisorption via π-stacking can be used for loading doxorubicin, a widely used cancer drug onto NGO functionalized with antibody for selective killing of cancer cells in vitro. Owing to its small size, intrinsic optical properties, large specific surface area, low cost, and useful non-covalent interactions with aromatic drug molecules, NGO is a promising new material for biological and medical applications. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. This article is published with open access at Springerlink.com     

A smart multifunctional nanocomposite for intracellular targeted drug delivery and self-release

A multifunctional 'all-in-one' nanocomposite is fabricated using a colloid, template and surface-modification method. This material encompasses magnetic induced target delivery, cell uptake promotion and controlled drug release in one system. The nanocomposite is characterized by scanning electron microscopy, transmission electron microscopy, x-ray diffraction, N(2) adsorption and vibrating sample magnetometry. The prepared material has a diameter of 350-400 nm, a high surface area of 420.29 m(2) g(-1), a pore size of 1.91 nm and a saturation magnetization of 32 emu g(-1). Doxorubicin (DOX) is loaded in mesopores and acid-sensitive blockers are introduced onto the orifices of the mesopores by a Schiff base linker to implement pH-dependent self-release. Folate was also introduced to improve DOX targeted delivery and endocytosis. The linkers remained intact to block pores with ferrocene valves and inhibit the diffusion of DOX at neutral pH. However, in lysosomes of cancer cells, which have a weak acidic pH, hydrolysis of the Schiff base group removes the nanovalves and allows the trapped DOX to be released. These processes are demonstrated by UV-visible absorption spectra, confocal fluorescence microscopy images and methyl thiazolyl tetrazolium assays in vitro, which suggest that the smart nanocomposite successfully integrates targeted drug delivery with internal stimulus induced self-release and is a potentially useful material for nanobiomedicine.     

Hollow manganese phosphate nanoparticles as smart multifunctional probes for cancer cell targeted magnetic resonance imaging and drug delivery

Multifunctional probes for simultaneous magnetic resonance imaging (MRI) and drug delivery have attracted considerable interest due to their promising potential applications in the early-stage diagnosis and therapy of the diseases. In this study, hollow manganese phosphate nanoparticles (HMP NPs) with an average diameter of 18 nm were synthesized and aminated through silanization, which enabled the covalent conjugation of biocompatible poly(ethylene glycol) (PEG) on their surfaces. The anti-tumor drug doxorubicin (DOX) could be loaded into the hollow cavities. Under physiological conditions (pH 7.4), the NPs showed low MRI T ₁ contrast (r ₁ = 1.19 L·mmol^?1·s^?1), whereas high T ₁ enhancement (r ₁ = 5.22 L·mmol^?1·s^?1) was achieved after dissolving them in endosome/lysosome mimetic conditions (pH 5.4). This is due to the fact that the NPs were easily eroded, which resulted in the release of Mn²⁺ at low pH. To use this interesting phenomenon for targeted DOX drug delivery, we conjugated the tumor-targeting ligand folic acid (FA) on HMP NPs and investigated their drug delivery capacity and cytotoxicity to cell lines expressing different amount of folate receptor (FR). KB cells showed more significant cellular uptake than HeLa cells and A549 cells, as confirmed by confocal laser scanning microscopy (CLSM), flow cytometry and cellular T ₁-weighted MRI. Furthermore, the drug-loaded HMP NPs exhibited greater cytotoxicity to KB cells. Our results suggest that functionalized HMP NPs can act as an effective multifunctional probe for selective diagnosis with MRI, as well as giving efficient targeted drug delivery.      

Acid-degradable gadolinium-based nanoscale coordination polymer: A potential platform for targeted drug delivery and potential magnetic resonance imaging

Nano Research - During conventional chemotherapy for cancer, nonspecific drug distribution, which causes serious side effects in normal tissues, is a serious limitation. Thus, it is desirable to...     

Golay pulse encoding for microbubble contrast imaging in ultrasound

We present a technique that uses Golay phase encoding, pulse inversion, and amplitude modulation (GPIAM) for microbubble contrast agent imaging with ultrasound. This technique improves the contrast-to-tissue ratio (CTR) by increasing the time-bandwidth product of the insonating waveforms. A nonlinear pulse compression algorithm is used to compress the signal energy upon receive. A 6.5-dB improvement in CTR was observed using an 8-chip GPIAM sequence compared to a conventional pulse-inversion amplitude-modulation sequence. The CTR improvement comes at the cost of a reduction in frame rate: GPIAM coding uses four input pulses whereas most contrast imaging sequences require two or three pulses. Our results showed that the microbubble response can be phase encoded and subsequently compressed using a nonlinear matched-filtering algorithm, in order to enhance the signal from the contrast agent, while maintaining resolution and suppressing the tissue signal.