Ratiometric Photoacoustic Signals Research Articles

Endogenous CO imaging in bacterial pneumonia with a NIR fluorescent probe

Bacterial pneumonia is a serious respiratory illness that poses a great threat to human life. Rapid and precise diagnosis of bacterial pneumonia is crucial for symptomatic clinical treatment. Endogenous carbon monoxide (CO) is regarded as a significant indicator of bacterial pneumonia; herein, we developed a near-infrared (NIR) probe for fluorescence and photoacoustic (PA) dual-mode imaging of endogenous CO in bacterial pneumonia. NO2-BODIPY could rapidly and specifically react with CO to produce strong NIR fluorescence as well as ratiometric PA signals. NO2-BODIPY has outstanding features including fast response, fluorescence/PA dual mode signals, good specificity, and a low limit of detection (LOD = 20.3 nM), which enables it to image endogenous CO in cells and bacterial pneumonia mice with high sensitivity and high contrast ratio. In particular, NO2-BODIPY has two-photon excited (1340 nm, σ1 = 1671 GM) NIR fluorescence and has been utilized to image endogenous CO in bacterial pneumonia mice with deep tissue penetration. NO2-BODIPY has been demonstrated a good capability of fluorescence/PA dual-mode imaging of CO in bacterial pneumonia mice, providing a precise manner to diagnose bacterial pneumonia.

PH-responsive ratiometric photoacoustic imaging of polyaniline nanoparticle-coated needle for targeted cancer biopsy

Cancer microenvironment exhibits lower pH compared to healthy tissues, a characteristic which can be exploited using a pH-responsive needle to increase the accuracy of cancer biopsy. A needle, coated with pH-responsive polyaniline (PANI) nanoparticles (PANI-needle), is developed for the minimally invasive and quantitative pH analysis of tissue based on ratiometric photoacoustic (PA) imaging. The ratiometric PA signal from the PANI-needle within the 850–700 nm wavelength range shows a linear response as pH changes from 7.5 to 6.5. Owing to the high surface area of nanostructured PANI, the PA signal of PANI-needle exhibits a fast and reversible response of less than a few seconds. In a tissue-mimicking hydrogel phantom composed of two regions with different pH, PA ratios of PANI-needle successfully differentiate the local pH. The PANI-needle coupled with ultrasound-guided PA imaging is a promising technology for detection of malignant tissue through quantitative pH analysis during needle biopsy.

A "Dual-Source, Dual-Activation" Strategy for an NIR-II Window Theranostic Nanosystem Enabling Optimal Photothermal-Ion Combination Therapy.

The activatable imaging technique in the second near-infrared window (NIR-II) utilizes the stimulation of cancer-associated biomarkers for specific imaging to guide precise NIR-II photothermal therapy. However, most activatable nanoprobes with single-source stimulation are insufficient in providing comprehensive information regarding the tumor, severely restricting the therapeutic optimization, especially in NIR-II photothermal therapy (PTT)-based combination therapy. Herein, a "dual-source, dual-activation" strategy-based multifunctional nanosystem, PPAC, is reported as a promising tool for activatable NIR-II fluorescence (FL)/ratiometric photoacoustic (PA) imaging-guided "localization-timing" photothermal-ion therapy (PTIT). A fibroblast activation protein (FAP)-responsive peptide to modify the surface of Pd nanosheets with excellent NIR-II absorption ability can efficiently cross-link BSA-CQ4T to realize NIR-II FL quenching, followed by the loading of Ag to construct the PPAC. Triggered by the specific cleavage with FAP on the perivascular cancer-associated fibroblasts (first source), the PPAC can correspondingly release BSA-CQ4T for rapid fluorescence recovery. The nanosystems are subsequently taken up by tumor cells, where the overexpressed H2 O2 (second source) promotes the oxidation of Ag shell to Ag+ , and further leads the real-time ratiometric PA signals (Ag-PA660/Pd-PA1050) that can monitor the Ag+ ions-related production efficiency and therapeutic performance. Intelligent integration of dual-modality imaging information can comprehensively provide the right time-point and site-specificity for selective NIR-II PTT.

Engineering a Ratiometric Photoacoustic Probe with a Hepatocyte-Specific Targeting Ability for Liver Injury Imaging.

In situ imaging of biological indicators is imperative for pathological research by utilizing an activatable photoacoustic (PA) probe. However, precise imaging in actual applications is hampered by the inevitable poor accumulation and low sensitivity. Herein, an amphiphilic molecular probe (AP) was rationally constructed as proof of concept for in situ imaging of drug-induced liver injury, which consists of a hydrophilic target unit and a superoxide anion radical (O2•-)-sensitive small-molecule PA moiety. The probe AP successfully realizes the selectivity and sensitivity toward O2•- in vitro and in living cells. Notably, the amphiphilic probe AP can be selectively retained in the liver and activated toward endogenous O2•- through receptor-mediated endocytosis inside hepatocytes. By virtue of the highly efficient accumulation at the liver, AP was further applied to assess isoniazid-induced liver injury through desired ratiometric PA signals. In addition, based on the fluctuation of O2•-, the therapeutic efficacy of hepatoprotective medicines for hepatotoxicity was analyzed in vivo. Therefore, the O2•--specific probe could serve as a promising molecular tool for early diagnosis study of other liver diseases and analysis of new potential therapeutic agents.

Efficient Polysulfide-Based Nanotheranostics for Triple-Negative Breast Cancer: Ratiometric Photoacoustics Monitored Tumor Microenvironment-Initiated H2 S Therapy.

The incidence of triple-negative breast cancer (TNBC) is difficult to predict, and TNBC has a high mortality rate among women worldwide. In this study, a theranostics approach is developed for TNBC with ratiometric photoacoustic monitored thiol-initiated hydrogen sulfide (H2 S) therapy. The ratiometric photoacoustic (PA) probe (CY) with a thiol-initiated H2 S donor (PSD) to form a nanosystem (CY-PSD nanoparticles) is integrated. In this theranostics approach, H2 S generated from PSD is sensed by CY based on ratiometric PA signals, which simultaneously pinpoints the tumor region. Additionally, H2 S is cytotoxic toward TNBC cells (MDA-MB 231), showing a tumor inhibition rate of 63%. To further verify its pharmacological mechanism, proteomics analysis is performed on tumors treated with CY-PSD nanoparticles. Cells are killed by the significant mitochondrial dysfunction via supressed energy supply and apoptosis initiation. Besides, the observed inhibition of oxidative stress also generates the cytotoxicity. Significant Kyoto Encyclopedia of Genes Genomes pathways related to TNBC are found to be inhibited. This H2 S theranostics approach updates the current anticancer therapies which brings promise for women suffering malignant breast cancer.

Design and Synthesis of a Ratiometric Photoacoustic Probe for In Situ Imaging of Zinc Ions in Deep Tissue In Vivo.

As a noninvasive deep-tissue imaging technique, photoacoustic (PA) imaging has great application potential in biomedicine and molecular diagnosis. The zinc ion (Zn2+), which is a necessary metal ion in the human body, plays a very important role in the regulation of gene transcription and metalloenzyme function. The imbalance of Zn2+ homeostasis is also associated with a variety of neurological diseases. Therefore, it is critically important to accurately image the steady-state changes of Zn2+ in vivo. However, no PA imaging method is currently available for Zn2+. To this end, we designed and synthesized the first PA probe of Zn2+, namely, CR-1 for in situ ratiometric imaging of Zn2+ in deep tissue in vivo. The CR-1molecule, combined with Zn2+, weakened the conjugation system of the π-electron in the CR-1 molecule, which resulted in the blue shift of its absorption peak from 710 nm to 532 nm. The PA signal intensity decreased at 710 nm and increased at 532 nm, and the ratiometric PA signal at these two wavelengths (PA532/PA710) showed a good linear relationship with the concentration of Zn2+ in the range of 0-50 μM, with a detection limit as low as 170 nM. Furthermore, this probe exhibits extremely fast responsiveness, is highly selective, and has excellent biocompatibility. We have used the developed PA probe for the ratiometric PA imaging of Zn2+ in the thigh tissue of mice, and we still can accurately image Zn2+ after covering chicken breast tissue on the surface of mice thigh. In light of these outstanding features, the developed PA probe has high potential for imaging Zn2+ in deep tissues; thus, it will open up new avenues for the study of the complex biochemical processes involving Zn2+ in vivo.

Synthesis and Self-Assembly Behavior of Chlorophyll Derivatives for Ratiometric Photoacoustic Signal Optimization.

Self-assembly provides researchers powerful tools for creating ordered functional structures and complex architectures. Investigation of in vivo self-assembly reveals the assembly/aggregation-induced retention (AIR) effect and enhanced targeting effect, which can be applied to promising biomedical applications by enhancing molecular accumulation in the target region. These unique bioeffects inspire the interest of researchers in construction of self-assembled nanomaterials in biological systems. Although many efforts have been achieved, the in-depth analysis of the relationship between assemblies and functions is rarely reported. Here, we focus on the relationship of chlorophyll-derivative assemblies and their photoacoustic signals and attempt to establish a method for monitoring the aggregation efficiency in vivo based on photoacoustic signals. Three arginine-rich peptide-purpurin molecules were designed and synthesized. The assembled capabilities and assembly processes of these molecules were characterized and monitored by UV, fluorescence, and CD spectra images of gradually changing polarities in mixed solvents, and the morphologies of the assemblies were observed by TEM. Furthermore, the relationship between the aggregation ratios of the molecules and the ratiometric photoacoustic signals was systemically studied. We prospect that the fundamental research in revealing objective laws will be useful for future guidance in optimizing photoacoustic detection windows and assembled molecule design.

Ratiometric Photoacoustic Nanoprobe for Bioimaging of Cu2.

Aberrant copper contents implicate numerous diseases including Alzheimer's disease and Wilson's disease. Conventional copper detection technologies are difficult to offer non-invasive and accurate deep tissue detection of copper. Here, we report a photoacoustic (PA) nanoprobe (NRh-IR-NMs) for ratiometric PA imaging of Cu2+. The nanoprobe consists of a selective Cu2+-responsive probe (NRh) as the indicator and a nonresponsive dye (IR) as the internal reference. In the presence of Cu2+, a selective Cu2+-induced structure change of NRh would take place, resulting in the increase of PA signal intensity increment at 716 nm (ΔPA716). However, the ΔPA834 which attributes to IR shows negligible change. Therefore, the ratiometric PA signal (ΔPA716/ΔPA834) could be used as an indicator for Cu2+ detection. This ratiometric PA detection method offers a noninvasive technology with high selectivity and tissue penetration depth, which is a promising tool for deep-tissue detection of Cu2+ in living organisms.

Activatable Semiconducting Theranostics: Simultaneous Generation and Ratiometric Photoacoustic Imaging of Reactive Oxygen Species In Vivo.

Enhancing the generation of reactive oxygen species (ROS) is an effective anticancer strategy. However, it is a great challenge to control the production and to image ROS in vivo, both of which are vital for improving the efficacy and accuracy of cancer therapy. Herein, an activatable semiconducting theranostic nanoparticle (NP) platform is developed that can simultaneously enhance ROS generation while self-monitoring its levels through ratiometric photoacoustic (PA) imaging. The NP platform can further guide in vivo therapeutic effect in tumors. The theranostic NP platform is composed of: (i) cisplatin prodrug and ferric ion catalyst for ROS generation, a part of combination cancer therapy; and (ii) a ratiometric PA imaging nanoprobe consisting of inert semiconducting perylene-diimide (PDI) and ROS activatable near-infrared dye (IR790s), used in ratiometric PA imaging of ROS during cancer treatment. Ratiometric PA signals are measured at two near-infrared excitation wavelengths: 680 and 790 nm for PDI and IR790s, respectively. The measurements show highly accurate visualization of • OH generation in vivo. This novel ROS responsive organic theranostic NP allows not only synergistic cancer chemotherapy but also real-time monitoring of the therapeutic effect through ratiometric PA imaging.

Ratiometric photoacoustic nanoprobes for monitoring and imaging of hydrogen sulfide in vivo.

Detection and visualization of hydrogen sulphide (H2S) is crucial for understanding its physiological and pathological roles towards human health and diseases, but precisely tracking of H2S in vivo remains challenging due to the limitations of available analytical methods. In this study, we developed a novel ratiometric photoacoustic (PA) nanoprobe for selective detection and imaging of H2S in biological fluids, live cells, brain tissues and animals. The nanoprobe AzHD-LP was fabricated by encapsulation of a newly synthesized H2S-responsive near-infrared (NIR) dye (AzHD) within a liposome (LP). The as-prepared AzHD-LP exhibits a dramatically red-shift response of its absorption peak after reduction reaction of AzHD with H2S: the absorbance of AzHD-LP centered at 600 and 700 nm is decreased and increased, respectively, producing a turn-on ratiometric PA signal in the presence of H2S. Typically, under the excitation of a 532 nm and 700 nm pulsed laser, the selective detection and imaging of H2S was achieved in aqueous solution, living cells and brain tissues of Alzheimer's diseased mice. Moreover, after AzHD-LP conjugated with a tumor-targeting peptide - c(RGDyK) as RGD-AzHD-LP - ratiometric PA mapping of the intratumoral generated H2S in the HCT116 colon tumor-bearing live mice was demonstrated.

Degradable Semiconducting Oligomer Amphiphile for Ratiometric Photoacoustic Imaging of Hypochlorite

Upregulation of highly reactive oxygen species (ROS) such as hypochlorite (ClO-) is associated with many pathological conditions including cardiovascular diseases, neuron degeneration, lung injury, and cancer. However, real-time imaging of ClO- is limited to the probes generally relying on fluorescence with shallow tissue-penetration depth. We here propose a self-assembly approach to develop activatable and degradable photoacoustic (PA) nanoprobes for in vivo imaging of ClO-. A near-infrared absorbing amphiphilic oligomer is synthesized to undergo degradation in the presence of a specific ROS (ClO-), which integrates a π-conjugated but ClO- oxidizable backbone with hydrophilic PEG side chains. This molecular architecture allows the oligomer to serve as a degradable nanocarrier to encapsulate the ROS-inert dye and self-assemble into structurally stable nanoparticles through both π-π stacking and hydrophobic interactions. The self-assembled nanoprobe exhibits sensitive and specific ratiometric PA signals toward ClO-, permitting ratiometric PA imaging of ClO- in the tumor of living mice.

In Situ Monitoring Intracellular Structural Change of Nanovehicles through Photoacoustic Signals Based on Phenylboronate-Linked RGD-Dextran/Purpurin 18 Conjugates.

The stimuli-responsive polymeric nanocarriers have been studied extensively, and their structural changes in cells are important for the controlled intracellular drug release. The present work reported RGD-dextran/purpurin 18 conjugates with pH-responsive phenylboronate as spacer for monitoring the structural change of nanovehicles through ratiometric photoacoustic (PA) signal. Phenylboronic acid modified purpurin 18 (NPBA-P18) could attach onto the RGD-decorated dextran (RGD-Dex), and the resulting RGD-Dex/NPBA-P18 (RDNP) conjugates with different molar ratios of RGD-Dex and NPBA-P18 were prepared. When the moles of NPBA-P18 were equivalent to more than triple of RGD-Dex, the single-stranded RDNP conjugates could self-assemble into nanoparticles in aqueous solution due to the fairly strong hydrophobicity of NPBA-P18. The pH-responsive aggregations of NPBA-P18 were investigated by UV-vis, fluorescence, and circular dichroism spectra, as well as transmission electron microscope. Based on distinct PA signals between monomeric and aggregated state, ratiometric PA signal of I750/I710 could be presented to trace the structural change progress. Compared with RDNP single chains, the nanoparticles exhibited effective cellular internalization through endocytosis pathway. Furthermore, the nanoparticles could form well-ordered aggregates responding to intracellular acidic environment, and the resulting structural change was also monitored by ratiometric PA signal. Therefore, the noninvasive PA approach could provide a deep insight into monitoring the intracellular structural change process of stimuli-responsive nanocarriers.

Ratiometric Photoacoustic Molecular Imaging for Methylmercury Detection in Living Subjects.

Photoacoustic molecular imaging is an emerging and promising diagnostic tool for heavy metal ions detection. Methylmercury (MeHg+ ) is one of the most potent neurotoxins, which damages the brain and nervous system of human beings through fish consumption. The development of a selective and sensitive method for MeHg+ detection is highly desirable. In this Communication, we develope a chemoselective photoacoustic sensor (LP-hCy7) composed of the liposome (LP) and MeHg+ -responsive near-infrared (NIR) cyanine dye (hCy7) for MeHg+ detection within living subjects, such as zebrafish and mouse. The as-prepared LP-hCy7 nanoprobe displays unique dual-shift NIR absorbance peaks and produces a normalized turn-on response after the reaction of MeHg+ and hCy7 through a mercury-promoted cyclization reaction. The absorbance intensities of LP-hCy7 nanoprobe at 690 and 860 nm are decreased and increased, respectively. The ratiometric photoacoustic signal (PA860/PA690) is noticeably increased in the presence of MeHg+ . These findings not only provide a ratiometric photoacoustic molecular imaging probe for the detection of metal ions in vivo, but also provides a tool for spectroscopic photoacoustic molecular imaging.

Quantitative Analysis of Caspase-1 Activity in Living Cells Through Dynamic Equilibrium of Chlorophyll-Based Nano-assembly Modulated Photoacoustic Signals

In situ construction of self-assemblies with unique property in living systems is a promising direction in the biomedical field. The noninvasive methods for significant enzyme activity in living cells or living subjects are imperative and meantime challenge tasks. The dynamic process of self-assembly of chlorophyll-based molecules in complex biological systems can be monitored by photoacoustic signals, which supports a noninvasive way to understand and quantitatively measure the activity of caspase-1. Furthermore, the activity of caspase-1 enables reflection of the bacterial infection in the early stage. Here, we present a biocompatible self-assembly from chlorophyll-peptide derivatives and first correlate the dynamic equilibrium with ratiometric photoacoustic signals. The intracellular equilibrium was managed by a bacterial infection precaution protein, i.e., caspase-1. This system offers a trial of noninvasive method to quantitative detection and real-time monitoring of bacterial infection in the early stage.