Photoacoustic Molecular Imaging Research Articles

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.

Nonlinearly enhanced photoacoustic microscopy by picosecond-laser-pumped excited state absorption of phthalocyanine nanoprobes

Efficient nanoprobes with unique optical properties are highly desirable for good-performance photoacoustic (PA) molecular imaging. The conventionally used PA nanoprobes basically take their ground-state absorption with contrast to the indelible wideband background absorption as the imaging mechanism, thus severely limiting the imaging contrast and sensitivity in practical applications. Herein, a nonlinearly enhanced PA microscopy mechanism with suppressed background interference and improved brightness has been proposed, by distinctively exploiting the picosecond-laser-pumped excited state absorption of the tin phthalocyanine (SnPc) nanoprobes that exhibit strong reverse saturable absorption below the laser damage threshold of tissues. Both theoretical simulation and experimental investigation have been performed to verify the nonlinearly enhanced optical and PA properties of the SnPc nanoprobes with comparison to conventional PA contrast agents. The enhanced PA imaging capability of the SnPc nanoprobes with improved sensitivity and contrast has been demonstrated by tissue-mimicking phantoms and in vivo mouse models. This work revolutionizes the traditional contrast mechanism of PA nanoprobes by introducing picosecond-laser-pumped nonlinear optical nanomaterials, which prefigures great potential for biosensing and bioimaging with improved contrast and sensitivity.

Technical validation studies of a dual-wavelength LED-based photoacoustic and ultrasound imaging system

Recent advances in high power, pulsed, light emitting diodes (LEDs) have shown potential as fast, robust and relatively inexpensive excitation sources for photoacoustic imaging (PAI), yet systematic characterization of performance for biomedical imaging is still lacking. We report here technical and biological validation studies of a commercial dual-wavelength LED-based PAI and ultrasound system. Phantoms and small animals were used to assess temporal precision. In phantom studies, we found high temporal stability of the LED-based PAI system, with no significant drift in performance observed during 6 h of operation or over 30 days of repeated measurements. In vivo dual-wavelength imaging was able to map the dynamics of changes in blood oxygenation during oxygen-enhanced imaging and reveal the kinetics of indocyanine green contrast agent inflow after intravenous administration (Tmax∼6 min). Taken together, these studies indicate that LED-based excitation could be promising for future application in functional and molecular PAI.

Photoacoustic molecular imaging-escorted adipose photodynamic-browning synergy for fighting obesity with virus-like complexes.

Photodynamic therapy and adipose browning induction are two promising approaches to reverse obesity. The former strategy acts rapidly and locally, whereas the latter has a more gradual and widespread effect. Despite their complementarity, they have rarely been combined and imaged non-invasively in vivo. Here we introduce an adipose-targeting hepatitis B core protein complex that contains a traceable photosensitizer (ZnPcS4 (zinc phthalocyanine tetrasulfonate)) and a browning agent (rosiglitazone) that allows simultaneous photodynamic and browning treatments, with photoacoustic molecular imaging. After intravenous injection in obese mice, the complex binds specifically to white adipose tissues, especially those rich in blood supply, and drives adipose reduction thanks to the synergy of ZnPcS4 photodynamics and rosiglitazone browning. Using photoacoustic molecular imaging, we could monitor the changes induced by the treatment, which included complex activity, lipid catabolism and angiogenesis. Our findings demonstrate the anti-obesity potential of our feedback-based synergic regimen orchestrated by the targeted hepatitis B core complex.

Photoacoustic Molecular Imaging for the Identification of Lymph Node Metastasis in Head and Neck Cancer Using an Anti-EGFR Antibody-Dye Conjugate.

The presence of lymph node (LN) metastases is an essential prognostic indicator in patients with head and neck squamous cell carcinoma (HNSCC). This study assessed photoacoustic molecular imaging (PAMI) of the antiepidermal growth factor receptor antibody (panitumumab) conjugated to a near-infrared fluorescent dye, IRDye800CW (panitumumab-IRDye800CW; pan800), for the identification of occult metastatic LNs in patients with HNSCC (n = 7). Methods: After in vitro photoacoustic imaging characterization of pan800, PAMI was performed on excised neck specimens from patients infused with pan800 before surgery. Freshly obtained neck specimens were imaged with 3-dimensional, multiwavelength spectroscopic PAMI (wavelengths of 680, 686, 740, 800, 860, 924, and 958 nm). Harvested LNs were then imaged with a closed-field near-infrared fluorescence imager and histologically examined by the pathologist to determine their metastatic status. Results: In total, 53 LNs with a maximum diameter of 10 mm were analyzed with photoacoustic and fluorescence imaging, of which 4 were determined to be metastatic on the final histopathologic report. Photoacoustic signals in the LNs corresponding to accumulated pan800 were spectrally unmixed using a linear least-square-error classification algorithm. The average thresholded photoacoustic signal intensity corresponding to pan800 was 5-fold higher for metastatic LNs than for benign LNs (2.50 ± 1.09 arbitrary units [a.u.] vs. 0.53 ± 0.32 a.u., P < 0.001). Fluorescence imaging showed that metastatic LNs had a 2-fold increase in fluorescence signal compared with benign LNs ex vivo (P < 0.01, 0.068 ± 0.027 a.u. vs. 0.035 ± 0.018 a.u.). Moreover, the ratio of the average of the highest 10% of the photoacoustic signal intensity over the total average, representative of the degree of heterogeneity in the pan800 signal in LNs, showed a significant difference between metastatic LNs and benign LNs (11.6 ± 13.4 vs. 1.8 ± 0.7, P < 0.01) and an area under the receiver-operating-characteristic curve of 0.96 (95% CI, 0.91-1.00). Conclusion: The data indicate that PAMI of IRDye800-labeled tumor-specific antibody may have the potential to identify occult LN metastasis perioperatively in HNSCC patients.

Tocilizumab-Conjugated Polymer Nanoparticles for NIR-II Photoacoustic-Imaging-Guided Therapy of Rheumatoid Arthritis.

The progressive debilitating nature of rheumatoid arthritis (RA) combined with its unknown etiology and initial similarity to other inflammatory diseases makes early diagnosis a significant challenge. Early recognition and treatment of RA is essential for achieving effective therapeutic outcome. NIR-II photoacoustic (PA) molecular imaging (PMI) is emerging as a promising new strategy for effective diagnosis and treatment guidance of RA, owing to its high sensitivity and specificity at large penetration depth. Herein, an antirheumatic targeted drug tocilizumab (TCZ) is conjugated to polymer nanoparticles (PNPs) to develop the first NIR-II theranostic nanoplatform, named TCZ-PNPs, for PA-imaging-guided therapy of RA. The TCZ-PNPs are demonstrated to have strong NIR-II extinction coefficient, high photostability and excellent biocompatibility. NIR-II PMI results reveal the excellent targeting abilities of TCZ-PNPs for the effective noninvasive diagnosis of RA joint tissue with a high signal-to noise ratio (SNR) of 35.8 dB in 3D PA tomography images. Remarkably, one-month treatment and PA monitoring using TCZ-PNPs shows RA is significantly suppressed. In addition, the therapeutic evaluation of RA mice by NIR-II PMI is shown to be consistent with clinical micro-CT and histological analysis. The TCZ-PNPs-assisted NIR-II PMI provides a new strategy for RA theranostics, therapeutic monitoring and the beyond.

Targeted imaging of orthotopic prostate cancer by using clinical transformable photoacoustic molecular probe

BackgroundTo obtain high-yield histological samples by targeted prostate cancer (PCa) biopsy is the current trend compared with transrectal ultrasound (TRUS)-guided systematic histological biopsy, which is regarded as the gold standard for prostate cancer (PCa) diagnosis. In this paper, we present a targeted PCa imaging strategy using a real-time molecular photoacoustic imaging system integrated with a handheld US probe (PAI/US) and synthesized an integrin αvβ3 targeted probe based on ICG (cRGD–ICG).MethodsTo prepare cRGD–ICG, ICG-NHS was linked to cRGD through carboxyl-co-reaction. In vitro PA imaging ability of cRGD–ICG was tested. Orthotopic PCa-bearing rats were used as animal models. After injected with either cRGD–ICG or non-targeted probe, rats were implemented with PA imaging to confirm the specific accumulation of cRGD–ICG at tumor region. Moreover, pathological frozen slices were made to observe distribution of the probe in prostate tissue ex vivo.ResultsA small molecular PAI probe was synthesized and exhibited excellent targeted imaging ability in vitro. In vivo photoacoustic imaging was carried out after intravenous injection of cRGD-ICG in orthotopic PCa-bearing rats under the facilitation of the PAI/US system. Maximum molecular photoacoustic signals were observed in the tumor area in vivo after the probe injection, which showed 3.8-fold higher signal enhancement than that in the control group (P < 0.05). Significantly higher cRGD-ICG accumulation was observed under confocal microscopy in the tumor region than in normal prostate tissue.ConclusionsAll our results showed that the comprehensive strategy provided a high-yield and reliable method for PCa diagnosis and targeted prostate biopsy, with great clinical translation potential.

Sound Out the Deep Colors: Photoacoustic Molecular Imaging at New Depths.

Photoacoustic tomography (PAT) has become increasingly popular for molecular imaging due to its unique optical absorption contrast, high spatial resolution, deep imaging depth, and high imaging speed. Yet, the strong optical attenuation of biological tissues has traditionally prevented PAT from penetrating more than a few centimeters and limited its application for studying deeply seated targets. A variety of PAT technologies have been developed to extend the imaging depth, including employing deep-penetrating microwaves and X-ray photons as excitation sources, delivering the light to the inside of the organ, reshaping the light wavefront to better focus into scattering medium, as well as improving the sensitivity of ultrasonic transducers. At the same time, novel optical fluence mapping algorithms and image reconstruction methods have been developed to improve the quantitative accuracy of PAT, which is crucial to recover weak molecular signals at larger depths. The development of highly-absorbing near-infrared PA molecular probes has also flourished to provide high sensitivity and specificity in studying cellular processes. This review aims to introduce the recent developments in deep PA molecular imaging, including novel imaging systems, image processing methods and molecular probes, as well as their representative biomedical applications. Existing challenges and future directions are also discussed.

Examining the technical feasibility of prostate cancer molecular imaging by transrectal photoacoustic tomography with transurethral illumination.

To pave the road toward clinical application of photoacoustic imaging in prostate cancer (PCa) diagnosis, we studied the technical feasibility and performance of transrectal photoacoustic (PA) imaging in mapping the indocyanine green (ICG) contrast agent, which is approved by FDA, in entire prostates by using light illumination via the urethral track. Experiments were conducted on a clinically relevant ex vivo model involving whole human prostates harvested from radical prostatectomy. The light source placed in the urethral track was an array of light emitting diodes (LEDs), illuminating the prostate with a delivered light power on the urethral wall within the safety limit. A dual-modality imaging system acquired PA and ultrasound (US) images simultaneously in the same way as in transrectal ultrasound (TRUS), with the US imaging presenting the tissue structure and PA imaging detecting the ICG solution. The imaging results demonstrated that tubes containing ICG solution at different concentrations can be detected at different positions in the prostate within a 2 cm range around from the urethral wall. Considering the sizes of regular human prostates, the proposed transurethral illumination in combination with transrectal US detection can facilitate PA molecular imaging over the entire prostate in a non-invasive manner, which makes it possible to further improve the PCa diagnosing efficiency with better molecular sensitivity and resulted better biopsy accuracy and much reduced pain for patients. Impact statement Differentiating cancerous tissues from healthy ones is critical in the diagnosis of prostate cancer (PCa). However, due to the low sensitivity of ultrasound (US) imaging to cancerous tissues, transrectal ultrasound (TRUS) guided biopsies, current standard procedure for diagnosing PCa, suffer from low core yield, leading to under-sampling and under-grading of clinically significant tumors. Via the experiment on the ex vivo human prostates, we evaluated the translational potential of photoacoustic imaging (PAI) based on a safe light emitting diodes (LED) source for detecting the molecular information in deep human prostate. We showed that transurethral light illumination in combination with transrectal US detection can facilitate PA molecular imaging over an entire human prostate in a non-invasive manner. The success of this study in the clinically relevant ex vivo human prostate model suggested a new strategy for PA and US combined imaging and detection of PCa.

An Activatable Cancer-Targeted Hydrogen Peroxide Probe for Photoacoustic and Fluorescence Imaging.

Reactive oxygen species play an important role in cancer, however, their promiscuous reactivity, low abundance, and short-lived nature limit our ability to study them in real time in living subjects with conventional noninvasive imaging methods. Photoacoustic imaging is an emerging modality for in vivo visualization of molecular processes with deep tissue penetration and high spatiotemporal resolution. Here, we describe the design and synthesis of a targeted, activatable probe for photoacoustic imaging, which is responsive to one of the major and abundant reactive oxygen species, hydrogen peroxide (H2O2). This bifunctional probe, which is also detectable with fluorescence imaging, is composed of a heptamethine carbocyanine dye scaffold for signal generation, a 2-deoxyglucose cancer localization moiety, and a boronic ester functionality that specifically detects and reacts to H2O2. The optical properties of the probe were characterized using absorption, fluorescence, and photoacoustic measurements; upon addition of pathophysiologic H2O2 concentrations, a clear increase in fluorescence and red-shift of the absorption and photoacoustic spectra were observed. Studies performed in vitro showed no significant toxicity and specific uptake of the probe into the cytosol in breast cancer cell lines. Importantly, intravenous injection of the probe led to targeted uptake and accumulation in solid tumors, which enabled noninvasive photoacoustic and fluorescence imaging of H2O2. In conclusion, the reported probe shows promise for the in vivo visualization of hydrogen peroxide. SIGNIFICANCE: This study presents the first activatable and cancer-targeted hydrogen peroxide probe for photoacoustic molecular imaging, paving the way for visualization of hydrogen peroxide at high spatiotemporal resolution in living subjects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5407/F1.large.jpg.

Enhanced Performance of a Molecular Photoacoustic Imaging Agent by Encapsulation in Mesoporous Silicon Nanoparticles.

Enhanced Performance of a Molecular Photoacoustic Imaging Agent by Encapsulation in Mesoporous Silicon Nanoparticles.

Molecular photoacoustic imaging with ultra-small gold nanoparticles.

Gold nanoparticles (AuNPs) below 10 nm in size can undergo renal clearance, which could facilitate their clinical translation. However, due to non-linear, direct relationship between their absorption and size, use of such "ultra-small" AuNPs as contrast agents for photoacoustic imaging (PAI) is challenging. This problem is complicated by the tendency of absorption for ultra-small AuNPs to be below the NIR range, which is optimal for in vivo imaging. Herein, we present 5-nm molecularly activated plasmonic nanosensors (MAPS) that produce a strong photoacoustic signal in labeled cancer cells in the NIR, demonstrating the feasibility of sensitive PAI with ultra-small AuNPs.

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.

Affibody-Indocyanine Green Based Contrast Agent for Photoacoustic and Fluorescence Molecular Imaging of B7-H3 Expression in Breast Cancer.

Spectroscopic photoacoustic (sPA) molecular imaging has high potential for identification of exogenous contrast agents targeted to specific markers. Antibody-dye conjugates have recently been used extensively for preclinical sPA and other optical imaging modalities for highly specific molecular imaging of breast cancer. However, antibody-based agents suffer from long circulation times that limit image specificity. Here, the efficacy of a small protein scaffold, the affibody (ABY), conjugated to indocyanine green (ICG), a near-infrared fluorescence dye, as a targeted molecular imaging probe is demonstrated. In particular, B7-H3 (CD276), a cellular receptor expressed in breast cancer, was imaged via sPA and fluorescence molecular imaging to differentiate invasive tumors from normal glands in mice. Administration of ICG conjugated to an ABY specific to B7-H3 (ABYB7-H3-ICG) showed significantly higher signal in mammary tumors compared to normal glands of mice. ABYB7-H3-ICG is a compelling scaffold for molecular sPA imaging for breast cancer detection.

Photoacoustic molecular imaging using combined acupuncture and gold nanorods as a composite contrast agent

In this study, we developed a novel photoacoustic imaging technique based on poly (ethyleneglycol)-coated (PEGylated) gold nanorods (PEG-GNRs) (as the contrast agent) combined with traditional Chinese medicine (TCM) acupuncture (as the auxiliary method) for quantitatively monitoring contrast enhancement in the vasculature of a mouse brain in vivo. This study takes advantage of the strong near-infrared absorption (peak at [Formula: see text][Formula: see text]nm) of GNRs and the ability to adjust the hemodynamics of acupuncture. Experimental results show that photoacoustic tomography (PAT) successfully reveals the optical absorption variation of the vasculature of the mouse brain in response to intravenous administration of GNRs and acupuncture at the Zusanli acupoint (ST36) both individually and combined. The quantitative measurement of contrast enhancement indicates that the composite contrast agents (integration of acupuncture and GNRs) would greatly enhance the photoacoustic imaging contrast. The quantitative results also have the potential to estimate the local concentration of GNRs and even the real-time effects of acupuncture.

Miniature gold nanorods for photoacoustic molecular imaging in the second near-infrared optical window.

In photoacoustic imaging, the second near infrared (NIR II) window is where tissue generates the least background signal. However, the large size of the few available contrast agents in this spectral range impedes their pharmacokinetics and decreases their thermal stability, leading to unreliable photoacoustic imaging. Here, we synthesize miniaturized gold nanorods absorbing in NIR II that are 5–11 times smaller than regular-sized gold nanorods with a similar aspect ratio. Under nanosecond pulsed laser illumination, small nanorods are about three times more thermally stable and generate 3.5 times stronger photoacoustic signal than absorption-matched large counterparts. These unexpected findings are confirmed using theoretical and numerical analysis, showing that photoacoustic signal is not only proportional to the optical absorption of the nanoparticle solution but also to the surface-to-volume ratio of the nanoparticles. In living tumor bearing mice, these small targeted nanorods display a 30% improvement in efficiency of agent delivery to tumors and generate 4.5 times greater photoacoustic contrast.

Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

Stimuli-Responsive Nanotheranostics for Real-Time Monitoring Drug Release by Photoacoustic Imaging.

Molecular photoacoustic imaging (PA) is a promising technology to understand tumor pathology and guide precision therapeutics. Despite the capability of activatable PA probes to image tumor-specific biomarkers, limitations in their molecular structure hamper them from effective drug delivery and the drug release monitoring. Herein, we developed a perylene diimide (PDI) based theranostic platform that provides noninvasive PA imaging signals to monitor tumor-specific pH-responsive drug release.Methods: we first designed and synthesized an acid-responsive amine-substituted PDI derivative. The pH sensitive properties of the PDI was demonstrated by density functional theory (DFT) calculations, UV-vis experiments and PA studies. The theranostic platform (THPDINs) was fabricated by self-assembly of the acid-responsive PDI, a pH irrelevant IR825 dye, and anti-cancer drug doxorubicin (DOX). The PA properties in various pH environment, drug delivery, cytotoxicity, cell uptake, ratiometric PA imaging and anti-tumor efficacy of the THPDINs were investigated in vitro and in vivo by using U87MG glioma cell line and U87MG tumor model.Results: We found that our designed PDI was sensitive to the tumor specific pH environment, reflected by absorbance shift, PA intensity and aggregation morphology changes in aqueous solution. The as-synthesized pH sensitive PDI acted as a molecular switch in the THPDINs, in which the switch can be triggered in the mild acidic tumor microenvironment to accelerate DOX release. Meanwhile, the DOX release could be monitored by ratiometric PA imaging.Conclusions: We developed a multifunctional PDI based theranostic platform for noninvasive real-time ratiometric PA imaging of tumor acidic pH and monitoring of drug release in living mice simultaneously. This strategy will shed light on the development of smart activatable theranostic nanoplatforms and will significantly advance the application of PA theranostics in biology and medicine.