Photoacoustic Molecular Imaging Research Articles

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.

Saliency enhancement method for photoacoustic molecular imaging based on Grüneisen relaxation nonlinear effect.

Photoacoustic molecular imaging technology has a wide range of applications in biomedical research. In practical scenarios, both the probes and blood generate signals, resulting in the saliency of the probes in the blood environment being diminished, impacting imaging quality. Although several methods have been proposed for saliency enhancement, they inevitably suffer from moderate generality and detection speed. The Grüneisen relaxation (GR) nonlinear effect offers an alternative for enhancing saliency and can improve generality and speed. In this article, the excitation and detection efficiencies are optimized to enhance the GR signal amplitude. Experimental studies show that the saliency of the probe is enhanced. Moreover, the issue of signal aliasing is studied to ensure the accuracy of enhancement results in the tissues. In a word, the feasibility of the GR-based imaging method in saliency enhancement is successfully demonstrated in the study, showing the superiorities of good generality and detection speed.

Second Near-Infrared Macrophage-Biomimetic Nanoprobes for Photoacoustic Imaging of Neuroinflammation.

Neuroinflammation is a significant pathological event involving the neurodegenerative process associated with many neurological disorders. Diagnosis and treatment of neuroinflammation in its early stage are essential for the prevention and management of neurological diseases. Herein, we designed macrophage membrane-coated photoacoustic (PA) probes (MSINPs), with targeting specificities based on naturally existing target-ligand interactions for the early diagnosis of neuroinflammation. The second near-infrared dye, IR1061, was doped into silica as the core and was encapsulated with a macrophage membrane. In vitro as well as in vivo, the MSINPs could target inflammatory cells via the inflammation chemotactic effect. PA imaging was used to trace the MSINPs in a neuroinflammation mouse model and showed a great targeted effect of MSINPs in the prefrontal cortex. Therefore, the biomimetic nanoprobe prepared in this study offers a new strategy for PA molecular imaging of neuroinflammation, which can enhance our understanding of the evolution of neuroinflammation in specific brain regions.

Thin flexible photoacoustic endoscopic probe with a distal-driven micro-step motor for pump-probe-based high-specific molecular imaging.

Conventional photoacoustic endoscopy (PAE) is mostly for structural imaging, and its molecular imaging ability is quite limited. In this work, we address this issue and present the development of a flexible acoustic-resolution-based photoacoustic endoscopic (AR-PAE) probe with an outer diameter of 8 mm. This probe is driven by a micro-step motor at the distal end, enabling flexible and precise angular step control to synchronize with the optical parametric oscillator (OPO) lasers. This probe retains the high spatial resolution, high penetration depth, and spectroscopic imaging ability of conventional AR-PAE. Moreover, it is capable for background-free high-specific photoacoustic molecular imaging with a novel pump-probe detection technique, as demonstrated by the distribution visualizing of the FDA approved contrast agent methylene blue (MB) in an ex-vivo pig ileum. This proposed method represents an important technical advancement in multimodal PAE, and can potentially make considerable contributions across various biomedical fields.

Piezocatalytic Schottky Junction Treats Atherosclerosis by a Biomimetic Trojan Horse Strategy.

The atherosclerotic vulnerable plaque is characterized by thefoamy macrophage burden, involving impaired cholesterol efflux and deficient efferocytosis. Correspondingly, piezocatalytic therapy is an emerging solution for eliminating the foamy macrophage burden with satisfactory spatiotemporal controllability and deep penetration depth. Herein, a biomimetic Trojan horse (Au-ZnO@MM) is engineered by coating the macrophage membrane (MM) onto the surface of a rod-like Au-ZnO Schottky Junction to effectively relieve the atherosclerotic progression. These Trojan horses with the coating of MM are actively transported into subsistent foamy macrophages and generate abundant reactive oxygen species (ROS) via ultrasound-activated piezocatalysis. ROS-initiated autophagy and mitochondrial dysfunction induce substantial cell apoptosis, alleviating the burden of subsistent foamy macrophages. The resulting apoptotic fragments further significantly facilitate cholesterol excretion and trigger efferocytosis of intraplaque fresh macrophages. Ultimately, the biomimetic Au-ZnO@MM piezocatalyst not only inhibits the foaming capacity of macrophages, but also improves the function of removing cell debris, which can stabilize atherosclerotic vulnerable plaque. Meanwhile, the plasmon resonance effect of integrated gold nanoparticles enables favorable photoacoustic molecular imaging for real-time image-guided atherosclerotic therapy. This proposed biomimetic Trojan horse strategy provides the paradigm of employing ultrasound-activated piezocatalytic methodology for enhanced atherosclerotic theranostics.

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Inflammation-Responsive Nanoagents for Activatable Photoacoustic Molecular Imaging and Tandem Therapies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) severely lowers the life quality by progressively destructing joint functions and eventually causing permanent disability, representing a pressing public health concern. The pathogenesis of RA includes the excessive production of proinflammatory cytokines and harmful oxygen-derived free radicals, such as nitric oxide (NO), which constitute vital targets for precise diagnosis and effective treatment of RA. In this study, we introduce an advanced nanoagent that integrates the RA microenvironment-activatable photoacoustic (PA) imaging with multitarget synergistic treatment for RA. A highly sensitive organic probe with NO-tunable energy transformation and molecular geometry is developed, which enables strong near-infrared absorption with a turn-on PA signal, and the active intramolecular motion could further boost PA conversion. The probe is coassembled with an inflammation-responsive prodrug to construct the theranostic nanoagent, on which a macrophage-derived cell membrane with natural tropism to the inflammatory sites is camouflaged to improve the targeting ability to inflamed joints. The nanoagent could not only sensitively detect RA and differentiate the severity but also efficiently alleviate RA symptoms and improve joint function. The combination of activatable probe-mediated NO scavenging and on-demand activation of anti-inflammatory prodrug significantly inhibits the proinflammatory factors and promotes macrophage repolarization from M1 to M2 phenotype. This meticulously designed nanoagent ingeniously integrates RA-specific PA molecular imaging with synergistic multitarget therapy, rendering tremendous promise for precise intervention of RA-related diseases.

In vivo characterization and analysis of glioblastoma at different stages using multiscale photoacoustic molecular imaging

Simultaneous spatio-temporal description of tumor microvasculature, blood-brain barrier, and immune activity is pivotal to understanding the evolution mechanisms of highly aggressive glioblastoma, one of the most common primary brain tumors in adults. However, the existing intravital imaging modalities are still difficult to achieve it in one step. Here, we present a dual-scale multi-wavelength photoacoustic imaging approach cooperative with/without unique optical dyes to overcome this dilemma. Label-free photoacoustic imaging depicted the multiple heterogeneous features of neovascularization in tumor progression. In combination with classic Evans blue assay, the microelectromechanical system based photoacoustic microscopy enabled dynamic quantification of BBB dysfunction. Concurrently, using self-fabricated targeted protein probe (αCD11b-HSA@A1094) for tumor-associated myeloid cells, unparalleled imaging contrast of cells infiltration associated with tumor progression was visualized by differential photoacoustic imaging in the second near-infrared window at dual scale. Our photoacoustic imaging approach has great potential for tumor-immune microenvironment visualization to systematically reveal the tumor infiltration, heterogeneity, and metastasis in intracranial tumors.

Optimizing Axial and Peripheral Substitutions in Si-Centered Naphthalocyanine Dyes for Enhancing Aqueous Solubility and Photoacoustic Signal Intensity.

Photoacoustic imaging using external contrast agents is emerging as a powerful modality for real-time molecular imaging of deep-seated tumors. There are several chromophores, such as indocyanine green and IRDye800, that can potentially be used for photoacoustic imaging; however, their use is limited due to several drawbacks, particularly photostability. There is, therefore, an urgent need to design agents to enhance contrast in photoacoustic imaging. Naphthalocyanine dyes have been demonstrated for their use as photoacoustic contrast agents; however, their low solubility in aqueous solvents and high aggregation propensity limit their application. In this study, we report the synthesis and characterization of silicon-centered naphthalocyanine dyes with high aqueous solubility and near infra-red (NIR) absorption in the range of 850-920 nm which make them ideal candidates for photoacoustic imaging. A series of Silicon-centered naphthalocyanine dyes were developed with varying axial and peripheral substitutions, all in an attempt to enhance their aqueous solubility and improve photophysical properties. We demonstrate that axial incorporation of charged ammonium mesylate group enhances water solubility. Moreover, the incorporation of peripheral 2-methoxyethoxy groups at the α-position modulates the electronic properties by altering the π-electron delocalization and enhancing photoacoustic signal amplitude. In addition, all the dyes were synthesized to incorporate an N-hydroxysuccinimidyl group to enable further bioconjugation. In summary, we report the synthesis of water-soluble silicon-centered naphthalocyanine dyes with a high photoacoustic signal amplitude that can potentially be used as contrast agents for molecular photoacoustic imaging.

Photoacoustic signal enhancement in dual-contrast gastrin-releasing peptide receptor-targeted nanobubbles.

Translatable imaging agents are a crucial element of successful molecular imaging. Photoacoustic molecular imaging relies on optical absorbing materials to generate a sufficient signal. However, few materials approved for human use can generate adequate photoacoustic responses. Here we report a new nanoengineering approach to further improve photoacoustic response from biocompatible materials. Our study shows that when optical absorbers are incorporated into the shell of a gaseous nanobubble, their photoacoustic signal can be significantly enhanced compared to the original form. As an example, we constructed nanobubbles using biocompatible indocyanine green (ICG) and biodegradable poly(lactic-co-glycolic acid) (PLGA). We demonstrated that these ICG nanobubbles generate a strong ultrasound signal and almost four-fold photoacoustic signal compared to the same concentration of ICG solution; our theoretical calculations corroborate this effect and elucidate the origin of the photoacoustic enhancement. To demonstrate their molecular imaging performance, we conjugated gastrin-releasing peptide receptor (GRPR) targeting ligands with the ICG nanobubbles. Our dual photoacoustic/ultrasound molecular imaging shows a more than three-fold enhancement in targeting specificity of the GRPR-targeted ICG nanobubbles, compared to untargeted nanobubbles or prostate cancer cells not expressing GRPR, in a prostate cancer xenograft mouse model in vivo.

In vivo three-dimensional multispectral photoacoustic imaging of dual enzyme-driven cyclic cascade reaction for tumor catalytic therapy

Non-invasive visualization of dynamic molecular events in real-time via molecular imaging may enable the monitoring of cascade catalytic reactions in living systems, however effective imaging modalities and a robust catalytic reaction system are lacking. Here we utilize three-dimensional (3D) multispectral photoacoustic (PA) molecular imaging to monitor in vivo cascade catalytic therapy based on a dual enzyme-driven cyclic reaction platform. The system consists of a two-dimensional (2D) Pd-based nanozyme conjugated with glucose oxidase (GOx). The combination of nanozyme and GOx can induce the PA signal variation of endogenous molecules. Combined with the PA response of the nanozyme, we can simultaneously map the 3D PA signals of dynamic endogenous and exogenous molecules associated with the catalytic process, thus providing a real-time non-invasive visualization. We can also treat tumors under the navigation of the PA imaging. Therefore, our study demonstrates the imaging-guided potential of 3D multispectral PA imaging in feedback-looped cascade catalytic therapy.

Detection of weak optical absorption by optical-resolution photoacoustic microscopy

Optical-resolution photoacoustic microscopy (OR-PAM) is one of the major implementations of photoacoustic (PA) imaging. With tightly focused optical illumination and high-frequency ultrasound detection, OR-PAM provides micrometer-level resolutions as well as high sensitivity to optical absorption contrast. Traditionally, it is assumed that the detected PA signal in OR-PAM has a linear dependence on the target’s optical absorption coefficient, which is the basis for quantitative functional and molecular PA imaging. In this paper, we demonstrate that, due to the limited detection bandwidth and detection view, OR-PAM can have a strong nonlinear dependence on the optical absorption, especially for weak optical absorption (<10 cm−1). We have investigated the nonlinear dependence in OR-PAM using numerical simulations, analyzed the underlining mechanisms, proposed potential solutions, and experimentally confirmed the results on phantoms. This work may correct a traditional misunderstanding of the OR-PAM signals and improve quantitative accuracy for functional and molecular applications.

Molecular Photoacoustic Imaging

Medicine has gone through several challenges to make it much more accurate and thus prolong the human being's life. A large part of this challenge is diseased, so early detection can help carry out treatment on time. There is a technology that allows detecting an abnormality within the body without using an invasive method. Ultrasound is a diagnostic test used to scan organs and tissues through sound waves. Although this technique has been widely used, the results are not desired because the images generated are not high resolution. On the other hand, X-rays are used because it presents an image with a much higher resolution than other techniques based on light waves or ultrasound; despite this, they are harmful to cells. In consequence of this problem, another method called molecular photoacoustic imaging has been implemented. This technique bridges the traditional depth limits of ballistic optical imaging and diffuse optical imaging's resolution limits, using the acoustic waves generated in response to laser light absorption, which has now shown potential for molecular imaging, allowing the visualization of biological processes in a non-invasive way. The purpose of this article is to give a critically scoped review of the physical, chemical, and biochemical characteristics of existing photoacoustic contrast agents, highlighting the pivotal applications and current challenges for molecular photoacoustic imaging.

Smart Nanozyme Platform with Activity‐Correlated Ratiometric Molecular Imaging for Predicting Therapeutic Effects

AbstractNanozymes with intrinsic enzyme‐like characteristics have attracted enormous research interest in biological application. However, there is a lack of facile approach for evaluating the catalytic activity of nanozymes in living system. Herein, we develop a novel manganese‐semiconducting polymer‐based nanozyme (MSPN) with oxidase‐like activity for reporting the catalytic activity of itself in acid‐induced cancer therapy via ratiometric near‐infrared fluorescence (NIRF)‐photoacoustic (PA) molecular imaging. Notably, MSPN possess oxidase‐like activity in tumor microenvironment, owing to the mixed‐valent MnOx nanoparticles, which can effectively kill cancer cells. Because the semiconducting polymer (PFODBT) is conjugated with oxidase‐responsive molecule (ORM), the catalytic activity of nanozyme can be correlated with the ratiometric signals of NIRF (FL695/FL825) and PA (PA680/PA780), which may provide new ideas for predicting anticancer efficacy of nanozymes in living system.

Smart Nanozyme Platform with Activity-Correlated Ratiometric Molecular Imaging for Predicting Therapeutic Effects.

Nanozymes with intrinsic enzyme-like characteristics have attracted enormous research interest in biological application. However, there is a lack of facile approach for evaluating the catalytic activity of nanozymes in living system. Herein, we develop a novel manganese-semiconducting polymer-based nanozyme (MSPN) with oxidase-like activity for reporting the catalytic activity of itself in acid-induced cancer therapy via ratiometric near-infrared fluorescence (NIRF)-photoacoustic (PA) molecular imaging. Notably, MSPN possess oxidase-like activity in tumor microenvironment, owing to the mixed-valent MnOx nanoparticles, which can effectively kill cancer cells. Because the semiconducting polymer (PFODBT) is conjugated with oxidase-responsive molecule (ORM), the catalytic activity of nanozyme can be correlated with the ratiometric signals of NIRF (FL695 /FL825 ) and PA (PA680 /PA780 ), which may provide new ideas for predicting anticancer efficacy of nanozymes in living system.

Copper-Catalyzed Covalent Dimerization of Near-Infrared Fluorescent Cyanine Dyes: Synergistic Enhancement of Photoacoustic Signals for Molecular Imaging of Tumors.

Photoacoustic (PA) imaging relies on the absorption of light by chromophores to generate acoustic waves used to delineate tissue structures and physiology. Here, we demonstrate that Cu(II) efficiently catalyzes the dimerization of diverse near-infrared (NIR) cyanine molecules, including a peptide conjugate. NMR spectroscopy revealed a C-C covalent bond along the heptamethine chains, creating stable molecules under conditions such as a wide range of solvents and pH mediums. Dimerization achieved >90% fluorescence quenching, enhanced photostability, and increased PA signals by a factor of about 4 at equimolar concentrations compared to the monomers. In vivo study with a mouse cancer model revealed that dimerization enhanced tumor retention and PA signal, allowing cancer detection at doses where the monomers are less effective. While the dye dimers highlighted peritumoral blood vessels, the PA signal for dimeric tumor-targeting dye-peptide conjugate, LS301, was diffuse throughout the entire tumor mass. A combination of the ease of synthesis, diversity of molecules that are amenable to Cu(II)-catalyzed dimerization, and the high acoustic wave amplification by these stable dimeric small molecules ushers a new strategy to develop clinically translatable PA molecular amplifiers for the emerging field of molecular photoacoustic imaging.

Evaluation of the Targeting and Therapeutic Efficiency of Anti-EGFR Functionalised Nanoparticles in Head and Neck Cancer Cells for Use in NIR-II Optical Window.

Gold nanoparticles have been indicated for use in a diagnostic and/or therapeutic role in several cancer types. The use of gold nanorods (AuNRs) with a surface plasmon resonance (SPR) in the second near-infrared II (NIR-II) optical window promises deeper anatomical penetration through increased maximum permissible exposure and lower optical attenuation. In this study, the targeting and therapeutic efficiency of anti-epidermal growth factor receptor (EGFR)-antibody-functionalised AuNRs with an SPR at 1064 nm was evaluated in vitro. Four cell lines, KYSE-30, CAL-27, Hep-G2 and MCF-7, which either over- or under-expressed EGFR, were used once confirmed by flow cytometry and immunofluorescence. Optical microscopy demonstrated a significant difference (p < 0.0001) between targeted AuNRs (tAuNRs) and untargeted AuNRs (uAuNRs) in all four cancer cell lines. This study demonstrated that anti-EGFR functionalisation significantly increased the association of tAuNRs with each EGFR-positive cancer cell. Considering this, the MTT assay showed that photothermal therapy (PTT) significantly increased cancer cell death (>97%) in head and neck cancer cell line CAL-27 using tAuNRs but not uAuNRs, apoptosis being the major mechanism of cell death. This successful targeting and therapeutic outcome highlight the future use of tAuNRs for molecular photoacoustic imaging or tumour treatment through plasmonic photothermal therapy.

Recent Progress on Molecular Photoacoustic Imaging with Carbon-Based Nanocomposites.

For biomedical imaging, the interest in noninvasive imaging methods is ever increasing. Among many modalities, photoacoustic imaging (PAI), which is a combination of optical and ultrasound imaging techniques, has received attention because of its unique advantages such as high spatial resolution, deep penetration, and safety. Incorporation of exogenous imaging agents further amplifies the effective value of PAI, since they can deliver other specified functions in addition to imaging. For these agents, carbon-based materials can show a large specific surface area and interesting optoelectronic properties, which increase their effectiveness and have proved their potential in providing a theragnostic platform (diagnosis + therapy) that is essential for clinical use. In this review, we introduce the current state of the PAI modality, address recent progress on PAI imaging that takes advantage of carbon-based agents, and offer a future perspective on advanced PAI systems using carbon-based agents.

Clinically translatable quantitative molecular photoacoustic imaging with liposome-encapsulated ICG J-aggregates

Photoacoustic (PA) imaging is a functional and molecular imaging technique capable of high sensitivity and spatiotemporal resolution at depth. Widespread use of PA imaging, however, is limited by currently available contrast agents, which either lack PA-signal-generation ability for deep imaging or their absorbance spectra overlap with hemoglobin, reducing sensitivity. Here we report on a PA contrast agent based on targeted liposomes loaded with J-aggregated indocyanine green (ICG) dye (i.e., PAtrace) that we synthesized, bioconjugated, and characterized to addresses these limitations. We then validated PAtrace in phantom, in vitro, and in vivo PA imaging environments for both spectral unmixing accuracy and targeting efficacy in a folate receptor alpha-positive ovarian cancer model. These study results show that PAtrace concurrently provides significantly improved contrast-agent quantification/sensitivity and SO2 estimation accuracy compared to monomeric ICG. PAtrace’s performance attributes and composition of FDA-approved components make it a promising agent for future clinical molecular PA imaging.

Activatable Carbocyanine Dimers for Photoacoustic and Fluorescent Detection of Protease Activity.

Activatable contrast agents are of ongoing research interest because they offer low background and high specificity to the imaging target. Engineered sensitivity to protease activity is particularly desirable because proteases are critical biomarkers in cancer, infectious disease, inflammatory disorders, and so forth. Herein, we developed and characterized a set of peptide-linked cyanine conjugates for dual-modal detection of protease activity via photoacoustic (PA) and fluorescence imaging. The peptide-dye conjugates were designed to undergo contact quenching via intramolecular dimerization and contained n dyes (n = 2, 3, or 4) with n - 1 cleavable peptide substrates. The absorption peaks of the conjugates were blue-shifted 50 nm relative to the free dye and had quenched fluorescence. This effect was sensitive to solvent polarity and could be reversed by solvent switching from water to dimethyl sulfoxide. Employing trypsin as a model protease, we observed a 2.5-fold recovery of the peak absorbance, 330-4600-fold fluorescent enhancement, and picomolar detection limits following proteolysis. The dimer probe was further characterized for PA activation. Proteolysis released single dye-peptide fragments that produced a 5-fold PA enhancement through the increased absorption at 680 nm with nanomolar sensitivity to trypsin. The peptide substrate could also be tuned for protease selectivity; as a proof-of-concept, we detected the main protease (Mpro) associated with the viral replication in SARS-CoV-2 infection. Last, the activated probe was imaged subcutaneously in mice and signal was linearly correlated to the cleaved probe. Overall, these results demonstrate a tunable scaffold for the PA molecular imaging of protease activity with potential value in areas such as disease monitoring, tumor imaging, intraoperative imaging, in vitro diagnostics, and point-of-care sensing.