Coupling Gold Nanospheres into Nanochain Constructs for High-Contrast, Longitudinal Photoacoustic Imaging.

Structural parameters play a crucial role in determining the electromagnetic and thermal responses of gold nanoconstructs (GNCs) at near-infrared (NIR) wavelengths. Therefore, developing GNCs for reliable, high-contrast photoacoustic imaging has been focused on adjusting structural parameters to achieve robust NIR light absorption with photostability. In this study, we introduce an efficient photoacoustic imaging contrast agent: gold sphere chains (GSCs) consisting of plasmonically coupled gold nanospheres. The chain geometry results in enhanced photoacoustic signal generation originating from outstanding photothermal characteristics compared to traditional gold contrast agents, such as gold nanorods. Furthermore, the GSCs produce consistent photoacoustic signals at laser fluences within the limits set by the American National Standards Institute. The exceptional photoacoustic response of GSCs allows for high-contrast photoacoustic imaging over multiple imaging sessions. Finally, we demonstrate the utility of our GSCs for molecular photoacoustic cancer imaging, both in vitro and in vivo, through the integration of a tumor-targeting moiety.

Hyper-Branched Gold Nanoconstructs for Photoacoustic Imaging in the Near-Infrared Optical Window.

In plasmonic nanoconstructs (NCs), fine-tuning interparticle interactions at the subnanoscale offer enhanced electromagnetic and thermal responses in the near-infrared (NIR) wavelength range. Due to tunable electromagnetic and thermal characteristics, NCs can be excellent photoacoustic (PA) imaging contrast agents. However, engineering plasmonic NCs that maximize light absorption efficiency across multiple polarization directions, i.e., exhibiting blackbody absorption behavior, remains challenging. Herein, we present the synthesis, computational simulation, and characterization of hyper-branched gold nanoconstructs (HBGNCs) as a highly efficient PA contrast agent. HBGNCs exhibit remarkable optical properties, including strong NIR absorption, high absorption efficiency across various polarization angles, and superior photostability compared to conventional standard plasmonic NC-based contrast agents such as gold nanorods and gold nanostars. In vitro and in vivo experiments confirm the suitability of HBGNCs for cancer imaging, showcasing their potential as reliable PA contrast agents and addressing the need for enhanced imaging contrast and stability in bioimaging applications.

In Vivo Photoacoustic Monitoring of Stem Cell Location and Apoptosis with Caspase-3-Responsive Nanosensors.

Stem cell therapy has immense potential in a variety of regenerative medicine applications. However, clinical stem cell therapy is severely limited by challenges in assessing the location and functional status of implanted cells in vivo. Thus, there is a great need for longitudinal, noninvasive stem cell monitoring. Here we introduce a multidisciplinary approach combining nanosensor-augmented stem cell labeling with ultrasound guided photoacoustic (US/PA) imaging for the spatial tracking and functional assessment of transplanted stem cell fate. Specifically, our nanosensor incorporates a peptide sequence that is selectively cleaved by caspase-3, the primary effector enzyme in mammalian cell apoptosis; this cleavage event causes labeled cells to show enhanced optical absorption in the first near-infrared (NIR) window. Optimization of labeling protocols and spectral characterization of the nanosensor in vitro showed a 2.4-fold increase in PA signal from labeled cells during apoptosis while simultaneously permitting cell localization. We then successfully tracked the location and apoptotic status of mesenchymal stem cells in a mouse hindlimb ischemia model for 2 weeks in vivo, demonstrating a 4.8-fold increase in PA signal and spectral slope changes in the first NIR window under proapoptotic (ischemic) conditions. We conclude that our nanosensor allows longitudinal, noninvasive, and nonionizing monitoring of stem cell location and apoptosis, which is a significant improvement over current end-point monitoring methods such as biopsies and histological staining of excised tissue.

Photoacoustic-guided ultrasound thermal imaging without prior knowledge of tissue composition.

Thermal strain imaging (TSI) is a widely investigated ultrasound (US) thermometry technique that is based on the temperature-dependent change in speed of sound. However, a major challenge of TSI is a calibration process to account for material-dependent thermal strain. In this study, we leverage nanoparticle (NP)-mediated photoacoustic (PA) thermometry to calibrate thermal strain and guide US thermal imaging. By controlling the molecular composition of the sub-micrometer layer surrounding the NPs, PA thermometry becomes independent of the thermal characteristics of the overall background tissue where the NPs reside. Thus accurate temperature measurements are obtainable from sparse NP-mediated PA signals. These measurements are used to guide TSI, allowing US thermometry to produce an expanded temperature map over the entire region of interest without prior knowledge of tissue composition. Our feasibility study in tissue-mimicking phantoms demonstrates the potential to improve TSI by integrating a PA-based calibration method that complements and guides US thermometry.

Tunable Interparticle Connectivity in Gold Nanosphere Assemblies for Efficient Photoacoustic Conversion.

Manipulating matter at the nanometer scale to create desired plasmonic nanostructures holds great promise in the field of biomedical photoacoustic (PA) imaging. We demonstrate a strategy for regulating PA signal generation from anisotropic nano-sized assemblies of gold nanospheres (Au NSs) by adjusting the inter-particle connectivity between neighboring Au NSs. The inter-particle connectivity is controlled by modulating the diameter and inter-particle spacing of Au NSs in the nanoassemblies. The results indicate that nanoassemblies with semi-connectivity, i.e., assemblies with a finite inter-particle spacing shorter than the theoretical limit of repulsion between nearby Au NSs, exhibit 3.4-fold and 2.4-fold higher PA signals compared to nanoassemblies with no connectivity and full connectivity, respectively. Furthermore, due to the reduced diffusion of Au atoms, the semi-connectivity Au nanoassemblies demonstrate high photodamage threshold and, therefore, excellent photostability at fluences above the current American National Standards Institute limits. The exceptional photostability of the semi-connectivity nanoassemblies highlights their potential to surpass conventional plasmonic contrast agents for continuing PA imaging. Collectively, our findings indicate that semi-connected nanostructures are a promising option for reliable, high-contrast PA imaging applications over multiple imaging sessions due to their strong PA signals and enhanced photostability.

Magnetic particles in motion: magneto-motive imaging and sensing.

Superparamagnetic nanoparticles have become an important tool in biomedicine. Their biocompatibility, controllable small size, and magnetic properties allow manipulation with an external magnetic field for a variety of diagnostic and therapeutic applications. Recently, the magnetically-induced motion of superparamagnetic nanoparticles has been investigated as a new source of imaging contrast. In magneto-motive imaging, an external, time-varying magnetic field is applied to move a magnetically labeled subject, such as labeled cells or tissue. Several major imaging modalities such as ultrasound, photoacoustic imaging, optical coherence tomography, and laser speckle tracking can utilize magneto-motive contrast to monitor biological events at smaller scales with enhanced contrast and sensitivity. In this review article, an overview of magneto-motive imaging techniques is presented, including synthesis of superparamagnetic nanoparticles, fundamental principles of magneto-motive force and its utility to excite labeled tissue within a viscoelastic medium, current capabilities of magneto-motive imaging modalities, and a discussion of the challenges and future outlook in the magneto-motive imaging domain.

Gold nanoparticles conjugated with DNA aptamer for photoacoustic detection of human matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) plays major roles in extracellular matrix (ECM) remodeling and membrane protein cleavage, suggesting a high correlation with cancer cell invasion and tumor metastasis. Here, we present a contrast agent based on a DNA aptamer that can selectively target human MMP-9 in the tumor microenvironment (TME) with high affinity and sensitivity. Surface modification of plasmonic gold nanospheres with the MMP-9 aptamer and its complementary sequences allows the nanospheres to aggregate in the presence of human MMP-9 through DNA displacement and hybridization. Aggregation of gold nanospheres enhances the optical absorption in the first near-infrared window (NIR-I) due to the plasmon coupling effect, thereby allowing us to detect the aggregated gold nanospheres within the TME via ultrasound-guided photoacoustic (US/PA) imaging. Selective and sensitive detection of human MMP-9 via US/PA imaging is demonstrated in solution of nanosensors with the pre-treatment of human MMP-9, in vitro in cell culture, and in vivo in a xenograft murine model of human breast cancer.

Bifunctional Janus Particles as Multivalent Synthetic Nanoparticle Antibodies (SNAbs) for Selective Depletion of Target Cells.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle Antibodies (SNAbs). SNAbs, with phage-display-identified cell-targeting ligands on one "face" and Fc-mimicking ligands on the opposite "face", were synthesized using a custom, multistep, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

A Trimodal Ultrasound, Photoacoustic and Magnetic Resonance Imaging Approach for Longitudinal Post-operative Monitoring of Stem Cells in the Spinal Cord.

Longitudinal monitoring of stem cells in the spinal cord could unveil critical information needed to understand regenerative processes, thereby expediting therapy development and translation. We introduce a post-operative trimodal imaging approach to monitor stem cells in the spinal cord over time. A key aspect of the approach is to label the stem cells with Prussian blue nanocubes (PBNCs), which simultaneously possess optical and magnetic properties for ultrasound-guided photoacoustic (US/PA) and magnetic resonance imaging (MRI) contrast. PBNC-Labeled stem cells were injected into the spinal cord of immunodeficient rats and tracked with US/PA imaging and MRI up to 14 d post-injection. Good agreement was observed between imaging modalities in vivo. Our results suggest that further development of the US/PA/MR imaging approach may create a powerful tool to aid development of regenerative therapies of the spinal cord, and the non-invasive imaging approach can ultimately be deployed in intra- and post-operative environments.

In vivo photoacoustic guidance of stem cell injection and delivery for regenerative spinal cord therapies.

Significance: Stem cell therapies are of interest for treating a variety of neurodegenerative diseases and injuries of the spinal cord. However, the lack of techniques for longitudinal monitoring of stem cell therapy progression is inhibiting clinical translation. Aim: The goal of this study is to demonstrate an intraoperative imaging approach to guide stem cell injection to the spinal cord in vivo. Results may ultimately support the development of an imaging tool that spans intra- or postoperative environments to guide therapy throughout treatment. Approach: Stem cells were labeled with Prussian blue nanocubes (PBNCs) to facilitate combined ultrasound and photoacoustic (US/PA) imaging to visualize stem cell injection and delivery to the spinal cord in vivo. US/PA results were confirmed by magnetic resonance imaging (MRI) and histology. Results: Real-time intraoperative US/PA image-guided injection of PBNC-labeled stem cells and three-dimensional volumetric images of injection provided feedback necessary for successful delivery of therapeutics into the spinal cord. Postoperative MRI confirmed delivery of PBNC-labeled stem cells. Conclusions: The nanoparticle-augmented US/PA approach successfully detected injection and delivery of stem cells into the spinal cord, confirmed by MRI. Our work demonstrated in vivo feasibility, which is a critical step toward the development of a US/PA/MRI platform to monitor regenerative spinal cord therapies.

Prussian blue nanocubes as a multimodal contrast agent for image-guided stem cell therapy of the spinal cord.

Translation of stem cell therapies to treat injuries and diseases of the spinal cord is hindered by lack of real-time monitoring techniques to guide regenerative therapies intra- and postoperatively. Thus, we developed an ultrasound (US), photoacoustic (PA), and magnetic resonance (MR) imaging approach augmented with Prussian blue nanocubes (PBNCs) to guide stem cell injections intraoperatively and monitor stem cell therapies in the spinal cord postoperatively. Per the clinical procedure, a multi-level laminectomy was performed in rats ex vivo, and PBNC-labeled stem cells were injected directly into the spinal cord while US/PA images were acquired. US/PA/MR images were also acquired post-surgery. Several features of the imaging approach were demonstrated including detection of low stem cell concentrations, real-time needle guidance and feedback on stem cell delivery, and good agreement between US/PA/MR images. These benefits span intra- and postoperative environments to support future development of this imaging tool.

Photomagnetic Prussian blue nanocubes: Synthesis, characterization, and biomedical applications.

Nanoparticles play an important role in biomedicine. We have developed a method for size-controlled synthesis of photomagnetic Prussian blue nanocubes (PBNCs) using superparamagnetic iron oxide nanoparticles (SPIONs) as precursors. The developed PBNCs have magnetic and optical properties desired in many biomedical diagnostic and therapeutic applications. Specifically, the size-tunable photomagnetic PBNCs exhibit high magnetic saturation, strong optical absorption with a peak at approximately 700 nm, and superior photostability. Our studies demonstrate that PBNCs can be used as MRI and photoacoustic imaging contrast agents in vivo. We also showed the utility of PBNCs for labeling and magnetic manipulation of cells. Dual magnetic and optical properties, together with excellent biocompatibility, render PBNCs an attractive contrast agent for both diagnostic and therapeutic applications. The use SPIONs as precursors for PBNCs provides flexibility and allows researchers to design theranostic agents according to required particle size, optical, and magnetic properties.

Effects of Freezing on Mesenchymal Stem Cells Labeled with Gold Nanoparticles.

Stem cell therapies are a promising treatment for many patients suffering from diseases with poor prognosis. However, clinical translation is inhibited by a lack of in vivo monitoring techniques to track stem cells throughout the course of treatment. Ultrasound-guided photoacoustic (PA) imaging of nanoparticle-labeled stem cells may be a solution. To allow PA tracking, stem cells must be labeled with an optically absorbing contrast agent. Gold nanoparticles are one option due to their cytocompatibility and strong optical absorption in the near-infrared region. However, stem cell labeling can require up to 24-h incubation with nanoparticles in culture before use. Although stem cell monitoring is critically needed, the additional preparation time may not be feasible-it is cost prohibitive and stem cell treatments should be readily available in emergency situations as well as scheduled procedures. To remedy this, stem cells can be labeled before freezing and long-term storage. While it is well known that stem cells retain their cellular function after freezing, storage, and thawing, the impact of gold nanoparticles on this process has yet to be investigated. Therefore, we assessed the viability, multipotency, and PA activity of gold nanosphere-labeled mesenchymal stem cells (MSCs) after freezing, storing, and thawing for 1 week, 1 month, or 2 months and compared to unlabeled, naive MSCs which were frozen, stored, and thawed at the same time points. Results indicated no substantial change in viability as assessed by the MTT assay. Differentiation, observed through adipogenesis and osteogenesis, was also comparable to controls. Finally, strong PA signals and similar PA spectral signatures remained. Further studies involving more diverse stem cell types and nanoparticles are required, but our data suggest that function and imaging properties of nanoparticle-labeled stem cells are maintained after freezing and storage, which improve translation of stem cell monitoring techniques by simplifying integration with clinical protocols. Impact statement Although stem cell tracking techniques are critically needed, stem cells must be labeled with contrast agents in advance of procedures, which is not clinically feasible due to increased procedure time. As a solution, a stock of labeled stem cells could be frozen and stored, ready for immediate use. Results showed that gold nanosphere-labeled stem cells can be frozen and stored long-term without impacting cellular function or photoacoustic imaging contrast, supporting further investigation of other contrast agents and cell types. Creating a bank of nanoparticle-labeled stem cells advances translation and scalability of stem cell tracking methods by improving integration with clinical protocols.

Development of a stem cell tracking platform for ophthalmic applications using ultrasound and photoacoustic imaging.

Glaucoma is the second leading cause of blindness in the world. Disease progression is associated with reduced cellularity in the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising therapy seeks to deliver stem cells to the TM to regenerate the tissue and restore its function. However, like many stem cell-based regenerative therapies, preclinical development relies heavily on histology to evaluate outcomes. To expedite clinical translation, we are developing an ultrasound/photoacoustic (US/PA) imaging platform for longitudinal tracking of stem cells in the anterior eye. Methods: Mesenchymal stem cells (MSCs) were labeled with gold nanospheres in vitro and injected through the cornea into the anterior chamber of ex vivo porcine eyes. Physiological pressure was imposed to mimic in vivo conditions. AuNS-labeled MSCs were injected through the cornea while single-wavelength US/PA images were acquired. At 5 hours post-injection, three-dimensional multi-wavelength US/PA datasets were acquired and spectroscopic analysis was used to detect AuNS-labeled MSCs. US/PA results were compared to fluorescent microscopy. Results: The US/PA imaging platform was able to provide real-time monitoring of the stem cell injection and distinguish AuNS-labeled MSCs from highly absorbing background tissues in the anterior segment. Conclusion: Our US/PA imaging approach can inform preclinical studies of stem cell therapies for glaucoma treatment, motivating further development of this theranostic imaging tool for ophthalmic applications.

Photoacoustic properties of anterior ocular tissues.

Clinical imaging techniques for the anterior segment of the eye provide excellent anatomical information, but molecular imaging techniques are lacking. Molecular photoacoustic imaging is one option to address this need, but implementation requires use of contrast agents to distinguish molecular targets from background photoacoustic signals. Contrast agents are typically selected based on a priori knowledge of photoacoustic properties of tissues. However, photoacoustic properties of anterior ocular tissues have not been studied yet. Herein, anterior segment anatomy and corresponding photoacoustic signals were analyzed in brown and blue porcine eyes ex vivo. Measured photoacoustic spectra were compared to known optical absorption spectra of endogenous chromophores. In general, experimentally measured photoacoustic spectra matched expectations based on absorption spectra of endogenous chromophores reported in the literature, and similar photoacoustic spectra were observed in blue and brown porcine eyes. However, unique light-tissue interactions at the iris modified photoacoustic signals from melanin. Finally, we demonstrated how the measured PA spectra established herein can be used for one application of molecular PA imaging, detecting photoacoustically labeled stem cells in the anterior segment for glaucoma treatment.

Photothermal transformation of Au-Ag nanocages under pulsed laser irradiation.

Pulsed laser irradiation has emerged as an effective means to photothermally transform plasmonic nanostructures after their use in different biomedical applications. However, the ability to predict the products after photothermal transformation requires extensive ex situ studies. Here, we report a systematic study of the photothermal transformation of Au-Ag nanocages with a localized surface plasmon resonance at ca. 750 nm under pulsed laser irradiation at different fluences and a pulse duration of 5 ns. At biologically relevant laser energies, the pulsed laser transforms Au-Ag nanocages into pseudo-spherical, solid nanoparticles. The solid nanoparticles contained similar numbers of Au and Ag atoms to the parent Au-Ag nanocages. At increased laser fluences (>16 mJ cm-2) and number of pulses (>150), the average diameter of the resulting pseudo-spherical particles increased due to the involvement of Ostwald ripening and/or attachment-based growth. The changes in optical properties as a result of the transformation were validated using simulations based on the discrete dipole approximation method, where the spectral profiles and peak positions of the initial and final states matched well with the experimentally derived data. The results may have implications for the future use of Au-Ag nanocages in biomedicine, catalysis, and sensing.

Photoacoustic Image-Guided Delivery of Plasmonic-Nanoparticle-Labeled Mesenchymal Stem Cells to the Spinal Cord.

Regenerative therapies using stem cells have great potential for treating neurodegenerative diseases and traumatic injuries in the spinal cord. In spite of significant research efforts, many therapies fail at the clinical phase. As stem cell technologies advance toward clinical use, there is a need for a minimally invasive, safe, affordable, and real-time imaging technique that allows for the accurate and safe monitoring of stem cell delivery in the operating room. In this work, we present a combined ultrasound and photoacoustic imaging tool to provide image-guided needle placement and monitoring of nanoparticle-labeled stem cell delivery into the spinal cord. We successfully tagged stem cells using gold nanospheres and provided image-guided delivery of stem cells into the spinal cord in real-time, detecting as few as 1000 cells. Ultrasound and photoacoustic imaging was used to guide needle placement for direct stem cell injection to minimize the risk of needle shear and accidental injury and to improve therapeutic outcomes with accurate, localized stem cell delivery. Following injections of various volumes of cells, three-dimensional ultrasound and photoacoustic images allowed the visualization of stem cell distribution along the spinal cord, showing the potential to monitor the migration of the cells in the future. The feasibility of quantitative imaging was also shown by correlating the total photoacoustic signal over the imaging volume to the volume of cells injected. Overall, the presented method may allow clinicians to utilize imaged-guided delivery for more accurate and safer stem cell delivery to the spinal cord.