KEYWORDS: Oxygen, Tumors, Photodynamic therapy, Nanodroplets, Image enhancement, Photoacoustic spectroscopy, Hypoxia, Solids, In vivo imaging, Real time imaging
This Conference Presentation, “Photoacoustic image guided oxygen enhanced photodynamic therapy of hypoxic tumors,” was recorded at SPIE Photonics West held in San Francisco, California, United States
This Conference Presentation, “Photoacoustic nanodroplets for oxygen enhanced photodynamic therapy,” was recorded for the Photonics West 2021 Digital Forum.
We introduce a preclinical imaging platform – a 3D photoacoustic/fluorescence tomography (PAFT) instrument augmented with an environmentally responsive dual-contrast biocompatible nanoprobe. The PAFT instrument was designed for simultaneous acquisition of photoacoustic and fluorescence orthogonal projections at each rotational position of a biological object, enabling direct co-registration of the two imaging modalities. The nanoprobe was based on liposomes loaded with J-aggregates of indocyanine green (PAtrace). Once PAtrace interacts with the environment, a transition from J-aggregate to monomeric ICG is induced. The subsequent recovery of monomeric ICG is characterized by dramatic changes in the optical absorption spectrum and reinstated fluorescence. In the activated state, PAtrace can be simultaneously detected by both imaging modes of the PAFT instrument using 780 nm excitation and fluorescence detection at 810 nm. The fluorescence imaging component is used to boost detection sensitivity by providing lowresolution map of activated nanoprobes, which are then more precisely mapped in 3D by the photoacoustic imaging component. Activated vs non-activated particles can be distinguished based on their different optical absorption peaks, removing the requirements for complex image registration between reference and detection scans. Preliminary phantom and in vivo animal imaging results showed successful activation and visualization of PAtrace with high sensitivity and resolution. The proposed PAFT-PAtrace imaging platform could be used in various functional and molecular imaging applications including multi-point in vivo assessment of early metastasis.
While molecular and cellular imaging can be used to visualize the conventional morphology characteristics of vulnerable plaques, there is a need to monitor other physiological factors correlated with high rupture rates; a high M1 activated macrophage concentration is one such indicator of high plaque vulnerability. Here, we present a molecularly targeted contrast agent for intravascular photoacoustic (IVPA) imaging consisting of liposomes loaded with indocyanine green (ICG) J-aggregates with high absorption at 890 nm, allowing for imaging in the presence of blood. This “Lipo-ICG” was targeted to a biomarker of M1 activated macrophages in vulnerable plaques: folate receptor beta (FRβ). The targeted liposomes accumulate in plaques through areas of endothelial dysfunction, while the liposome encapsulation prevents nonspecific interaction with lipids and endothelium. Lipo-ICG specifically interacts with M1 activated macrophages, causing a spectral shift and change in the 890/780 nm photoacoustic intensity ratio upon breakdown of J-aggregates. This sensing mechanism enables assessment of the M1 activated macrophage concentration, providing a measure of plaque vulnerability. In a pilot in vivo study utilizing ApoE deficient mouse models of atherosclerosis, diseased mice showed increased uptake of FRβ targeted Lipo-ICG in the heart and arteries vs. normal mice. Likewise, targeted Lipo-ICG showed increased uptake vs. two non-targeted controls. Thus, we successfully synthesized a contrast agent to detect M1 activated macrophages in high risk atherosclerotic plaques and exhibited targeting both in vitro and in vivo. This biocompatible agent could enable M1 macrophage detection, allowing better clinical decision making in treatment of atherosclerosis.
The ability to image metallic implants is important for medical applications ranging from diagnosis to therapy. Photoacoustic (PA) imaging has been recently pursued as a means to localize metallic implants in soft tissue. The work presented herein investigates different mechanisms to modulate the PA signal generated by macroscopic metallic surfaces. Wires of five different metals are tested to simulate medical implants/tools, while surface roughness is altered or physical vapor deposition (PVD) coatings are added to change the wires’ overall optical absorption. PA imaging data of the wires are acquired at 970 nm. Results indicate that PA signal generation predominately occurs in a wire’s metallic surface and not its aqueous surroundings. PA signal generation is similar for all metals tested, while addition of PVD coatings offers significant modulations (i.e., 4-dB enhancement and 26-dB reduction achieved) in PA signal generation. Results also suggest that PA signal increases with increasing surface roughness. Different coating and roughness schemes are then successfully utilized to generate spatial PA signal patterns. This work demonstrates the potential of surface modifications to enhance or reduce PA signal generation to permit improved PA imaging of implants/tools (i.e., providing location/orientation information) or to allow PA imaging of surrounding tissue.
The use of near-infrared wavelengths for photoacoustic (PA) imaging takes advantage of the relatively low
inherent absorption of tissues and has encouraged the development of agents which show high contrast in
this range. Here, we describe the modification of a commercially available PA imaging system (Vevo
LAZR, VisualSonics, Toronto) to take advantage of the 532nm and 1064nm wavelengths inherent in the
generation of the currently tuneable range of 680 to 970nm and in the use of these two wavelengths to
assess contrast agents.
The photoacoustic imaging system generated light from a Nd/YAG laser modified to extract the 532 and
1064nm wavelengths in addition to its OPO-derived tuneable range (680 - 970 nm) and deliver this light
through a fiber integrated into a linear array transducer (LZ400, VisualSonics).
Gold nanorods (UT Austin), carbon nanotubes (Stanford U), DyLight 550 (Thermo Fisher) and blood were
imaged in a phantom (PE20 tubing) and in a hindlimb subcutaneous tumor in vivo to determine their
photoacoustic signal intensity at all wavelengths.
In the phantom and in vivo, all agents caused an enhancement of the photoacoustic signal at their respective
peak absorbance wavelengths. These results show that the 532nm and 1064nm wavelengths could prove
useful in biomedical imaging due to the contrast agents customized for them. The 1064nm wavelength in
particular has the advantage of having very low generation of endogenous signal in vivo, making agents
tuned to this wavelength ideal for targeted contrast imaging.
Delivery of contrast agents and their interaction with cells is emerging as an important tool in cancer imaging and
therapy. An alternative to traditional molecular targeting schemes that induce endocytotic uptake of contrast agents in
cells is presented here. Specifically, the application of high-intensity, focused ultrasound (HIFU) was used to enhance
uptake of gold nanorods in pancreatic cancer cells in vitro. A significant increase was observed in gold nanorod uptake
when cells were incubated with nanorods and treated with HIFU. Additionally, inclusion of liquid-filled, perfluorocarbon
(PFC) microdroplets in cell samples incubated with nanorods and treated with HIFU exhibited greater uptake of gold
over those samples exposed to HIFU without microdroplets. Furthermore, the level of acoustic pressure required to
increase nanoparticle uptake did not significantly decrease cell viability. Therefore, improved intracellular delivery of
nanoparticle contrast agents is possible using HIFU without compromising cell viability. Since nanoparticle delivery is
mechanically induced, this method can apply to a broad range of cancer imaging and therapy applications.
Due to their optical absorption properties, metallic nanoparticles are excellent photoacoustic imaging contrast agents. A silver nanosystem is presented here as a potential contrast agent for photoacoustic imaging and image-guided therapy. Currently, the nanosystem consists of a porous silver layer deposited on the surface of spherical silica cores ranging in diameter from 180 to 520 nm. The porous nature of the silver layer will allow for release of drugs or other therapeutic agents encapsulated in the core in future applications. In their current PEGylated form, the silver nanosystem is shown to be nontoxic in vitro at concentrations of silver up to 2 mg/ml. Furthermore, the near-infrared absorbance properties of the nanosystem are demonstrated by measuring strong, concentration-dependent photoacoustic signal from the silver nanosystem embedded in an ex vivo tissue sample. Our study suggests that silver nanosystems can be used as multifunctional agents capable of augmenting image-guided therapy techniques.
The complementary information provided by the ultrasound and photoacoustic imaging modalities has sparked their use
in combination in recent years. We introduce a dual contrast agent capable of providing image contrast enhancement for
both modalities simultaneously. The dual contrast agent is a small liquid perfluorocarbon droplet encased with bovine
serum albumin and loaded with plasmonic nanoparticles. The plasmonic metal nanoparticles themselves act as a
photoacoustic contrast agent. Furthermore, the perfluorocarbon droplet creates acoustic impedance mismatch between
itself and the surrounding tissue, allowing it to act as an ultrasound contrast agent. Experiments demonstrating the
performance of this dual contrast agent in ultrasound and photoacoustic imaging are presented. Differences in contrast
mechanisms of the dual agent are highlighted, and finally, the applications for the agent are discussed.
Ultrasound imaging can provide excellent resolution at reasonable depths while retaining the advantages of being nonionizing,
cost-effective and portable. However, the contrast in ultrasound imaging is limited, and various ultrasoundbased
techniques such as photoacoustic (PA) and magneto-motive ultrasound (MMUS) imaging have been developed to
augment ultrasound imaging. Photoacoustic imaging enhances imaging contrast by visualizing the optical absorption of
either tissue or injected contrast agents (e.g., gold or silver nanoparticles). MMUS imaging enhances the sensitivity and
specificity of ultrasound based on the detection of magnetic nanoparticles perturbed by an external magnetic field. This
paper presents integrated magneto-photo-acoustic (MPA) imaging - a fusion of complementary ultrasound-based
imaging techniques. To demonstrate the feasibility of MPA imaging, porcine ex-vivo tissue experiments were performed
using a dual contrast (magnetic/plasmonic) agent. Spatially
co-registered and temporally consecutive ultrasound,
photoacoustic, and magneto-motive ultrasound images of the same
cross-section of tissue were obtained. Our ex-vivo
results indicate that magneto-photo-acoustic imaging can be used to detect magnetic/plasmonic nanoparticles with high
resolution, sensitivity and contrast. Therefore, our study suggests that magneto-photo-acoustic images can identify the
morphological properties, molecular information and complementary functional information of the tissue.
Image-guided molecular photothermal therapy using targeted gold nanoparticles acting as photoabsorbers can be used to
noninvasively treat various medical conditions including cancer. Among different types of gold nanoparticles, gold
nanorods are an attractive candidate for both photothermal therapy and photoacoustic imaging due to their high and
tunable optical absorption cross-section. However, nanorods are not thermodynamically stable; under laser exposure, the
nanorods can easily transform to spheres, thus changing their desired optical properties. In this study, gold-silica coreshell
nanorods were prepared by coating silica directly onto the surface of PEGylated gold nanorods using a modified
Stöber method. The nanorods were exposed to 800 nm wavelength, 7 ns pulses of light at a 10 Hz pulse repetition rate.
For different fluences ranging from 0 to 8 mJ/cm2, the optical extinction spectrum was measured before and after the
exposure to investigate their photothermal stability. Finally, the effectiveness of gold-silica core-shell nanoparticles as a
photoacoustic contrast agent and photothermal nanoabsorber was tested using inclusion-embedded phantoms and a
combined ultrasound and photoacoustic imaging system. The results of our study suggest that gold-silica core-shell
nanorods are excellent candidates for image-guided molecular photothermal therapy.
A dual contrast agent that combines perfluorocarbon droplets and metal nanoparticles has been developed for combined
ultrasound and photoacoustic imaging. Metal nanoparticles were incorporated in dodecafluoropentane (DDFP) droplets
encapsulated in a bovine serum albumin (BSA) shell. To embed aqueous colloidal suspensions of metal nanoparticles in
DDFP, a phase transfer of the plasmonic nanoparticles was completed in two different strategies, a single and a double
ligand exchange of capping materials. Emulsion techniques were used to encapsulate phase transferred metal
nanoparticles within the DDFP droplets. Spectrophotometry and cryogenic transmission electron microscopy were used
to characterize and to confirm successful fabrication of the dual contrast agent.
We have developed a multifunctional nanoagent, termed the combined imaging and therapy
nanocage system (CIT-NS). This nanosystem platform consists of a poly(lactic-co-glycolic) acid polymer
core and outer silver cage network. The inner core of CIT-NS is capable of carrying drugs, such as the
chemotherapeutic agent doxorubicin, or imaging contrast agents, such as dyes or fluorescent compounds.
The outer silver cage is specifically designed to enhance contrast in photoacoustic imaging, i.e., acoustic
imaging of optical absorption. In this paper, methods to create the CIT-NS are described. Initial
characterization indicates that the developed CIT-NS will significantly increase contrast in photoacoustic
imaging while retaining the potential to deliver large payloads of drug simultaneously. Therefore, the CITNS
may enable multi-modal imaging approaches or simultaneous imaging and therapy strategies to
improve treatment and detection of cancer and other pathologies.
A new metallodielectric nanoparticle consisting of a silica core and silver outer cage was
developed for the purpose of enhancing photoacoustic imaging contrast in pancreatic tissue. These
nanocages were injected into an ex vivo porcine pancreas and imaged using a combined photoacoustic and
ultrasound (PAUS) assembly. This custom-designed PAUS assembly delivered 800 nm light through a
fiber optical light delivery system integrated with 128 element linear array transducer operating at 7.5 MHz
center frequency. Imaging results prove that the nanocage contrast agents have the ability to enhance
photoacoustic imaging contrast. Furthermore, the value of the combined PAUS imaging modality was
demonstrated as the location of nanocages against background native tissue was evident. Future
applications of these nanocage contrast agents could include targeting them to pancreatic cancer for
enhancement of photoacoustic imaging for diagnosis and therapy.
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