KEYWORDS: Photoacoustic spectroscopy, Molecules, Absorption, Ultrasonography, Photoacoustic imaging, In vivo imaging, Signal detection, Tissues, Waveguides, In vitro testing
Activatable photoacoustic probes hold great promise for in vivo imaging of enzyme activity as they exhibit high contrast
and high selectivity at depths similar to that of ultrasound imaging. Here we report the synthesis and testing of a matrix
metalloproteinase 2 (MMP-2) specific peptide probe capable of changing its transient photoacoustic lifetime from short
to long after activation. The intact probe comprises an enzyme-specific cleavable sequence conjugated to a pair of
methylene blue (MB) molecules that dimerize due to forced proximity, resulting in static quenching. Upon cleavage, the
MB molecules dissociate and recover their intrinsic excited-state lifetime of more than 2 μs. We demonstrated using a
pump-probe photoacoustic imaging approach that the cleaved probe exhibits a transient signal with lifetime comparable
to that of MB monomers, whereas no lifetime was detected for the intact probe. This could allow for detection of the
cleaved probe in the presence of high levels of intact probe as well as short transients due to endogenous tissue
absorbers, thereby providing high contrast and low background noise. Finally, we have compared peptide sequences of
varying length and structure using absorption spectrometry in order to select the probe best suited for our imaging needs.
Our results suggest that both factors could potentially have an impact on dimerization efficiency and the cleavage rate of
the peptide. However, all probes provided a high degree of dimerization and efficient separation after cleavage,
indicating that a lifetime-based activatable peptide can be constructed for in vivo applications using a two-wavelength
imaging approach.
The oxygen partial pressure (pO2), which results from the balance between oxygen delivery and its consumption, is a key component of the physiological state of a tissue. Images of oxygen distribution can provide essential information for identifying hypoxic tissue and optimizing cancer treatment. Previously, we have reported a noninvasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor on small animals to identify hypoxia area. We also showed that PALI is able monitor changes of tissue oxygen, in an acute ischemia and breathing modulation model. Here we present our work on developing a treatment/imaging modality (PDT-PALI) that integrates PDT and a combined ultrasound/photoacoustic imaging system. The system provides real-time feedback of three essential parameters namely: tissue oxygen, light penetration in tumor location, and distribution of photosensitizer. Tissue oxygen imaging is performed by applying PALI, which relies on photoacoustic probing of oxygen-dependent, excitation lifetime of Methylene Blue (MB) photosensitizer. Lifetime information can also be used to generate image showing the distribution of photosensitizer. The level and penetration depth of PDT illumination can be deduced from photoacoustic imaging at the same wavelength. All images will be combined with ultrasound B-mode images for anatomical reference.
Activatable photoacoustic probes have a promising future due to their ability to provide high-resolution, high-penetration depth information on enzyme activity in vivo. Spectral identification methods, however, suffer from heterogeneous optical properties and wavelength-dependent light attenuation in tissue, thereby limiting the effective suppression of background noise signal. Our approach is predicated on probing the excited-state lifetime of a dual-labeled methylene blue (MB) probe that changes its lifetime from short to long upon cleavage. Recently, we have reported on the ability of our system to probe the long triplet lifetime of free MB monomers in solution and to differentiate between monomers and dimers based on their lifetime contrast. Here we introduce an improvement to our system that significantly increases the system sensitivity to fast changes, and reduces the minimum resolvable lifetime down to a few nanoseconds. We applied this method to probe the excited-state lifetime of a covalently coupled dual methylene blue-lysine conjugate (MB2K) in a mixed MB/MB2K solution. Preliminary results show that a stable dimeric bond is formed between the chromophores within the conjugate, and that this conjugate is statically quenched. Examination of the transient absorption of MB2K reveals it does not exhibit a triplet excited-state lifetime, suggesting that it undergoes a fast deexcitation process directly from the singlet state. Finally, we demonstrate how the transient photoacoustic lifetime signal can be used to selectively detect the presence of MB monomers while improving background noise suppression by differentiating the lifetime of free MB dye with other absorbing structures.
Tissue oxygen plays a critical role in maintaining tissue viability and in various diseases, including response to therapy. Images of oxygen distribution provide the history of tissue hypoxia and evidence of oxygen availability in the circulatory system. Currently available methods of direct measuring or imaging tissue oxygen all have significant limitations. Previously, we have reported a non-invasive in vivo imaging modality based on photoacoustic lifetime. The technique maps the excited triplet state of oxygen-sensitive dye, thus reflects the spatial and temporal distribution of tissue oxygen. We have applied PALI on tumor hypoxia in small animals, and the hypoxic region imaged by PALI is consistent with the site of the tumor imaged by ultrasound. Here, we present two studies of applying PALI to monitor changes of tissue oxygen by modulations. The first study involves an acute ischemia model using a thin thread tied around the hind limb of a normal mouse to reduce the blood flow. PALI images were acquired before, during, and after the restriction. The drop of muscle pO2 and recovery from hypoxia due to reperfusion were observed by PALI tracking the same region. The second study modulates tissue oxygen by controlling the percentage of oxygen the mouse inhales. We demonstrate that PALI is able to reflect the change of oxygen level with respect to both hyperbaric and hypobaric conditions. We expect this technique to be very attractive for a range of clinical applications in which tissue oxygen mapping would improve therapy decision making and treatment planning.
Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO 2 ) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO 2 -dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO 2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO 2 was consistently detected by both methods in tumors.
Activatable photoacoustic probes efficiently combine the high spatial resolution and penetration depth of ultrasound with the high optical contrast and versatility of molecular imaging agents. Our approach is based on photoacoustic probing of the excited-state lifetime of methylene blue (MB), a fluorophore widely used in clinical therapeutic and diagnostic applications. Upon aggregation, static quenching between the bound molecules dramatically shortens their lifetime by three orders of magnitude. We present preliminary results demonstrating the ability of photoacoustic imaging to probe the lifetime contrast between monomers and dimers with high sensitivity in cylindrical phantoms. Gradual dimerization enhancement, driven by the addition of increasing concentrations of sodium sulfate to a MB solution, showed that lifetime-based photoacoustic probing decreases linearly with monomer concentration. Similarly, the addition of 4 mM sodium dodecyl sulfate, a concentration that amplifies MB aggregation and reduces the monomer concentration by more than 20-fold, led to a signal decrease of more than 20 dB compared to a solution free of surfactant. These results suggest that photoacoustic imaging can be used to selectively detect the presence of monomers. We conclude by discussing the implementation of the monomer–dimer contrast mechanism for the development of an enzyme-specific activatable probe.
Oxygen plays a key role in the energy metabolism of living organisms. Any imbalance in the oxygen levels will affect
the metabolic homeostasis and lead to pathophysiological diseases. Hypoxia, a status of low tissue oxygen, is a key
factor in tumor biology as it is highly prominent in tumor tissues. However, clinical tools for assessing tissue
oxygenation are limited. The gold standard is polarographic needle electrode which is invasive and not capable of
mapping (imaging) the oxygen content in tissue.
We applied the method of photoacoustic lifetime imaging (PALI) of oxygen-sensitive dye to small animal tissue hypoxia
research. PALI is new technology for direct, non-invasive imaging of oxygen. The technique is based on mapping the
oxygen-dependent transient optical absorption of Methylene Blue (MB) by pump-probe photoacoustic imaging. Our
studies show the feasibility of imaging of dissolved oxygen distribution in phantoms. In vivo experiments demonstrate
that the hypoxia region is consistent with the site of subcutaneously xenografted prostate tumor in mice with adequate
spatial resolution and penetration depth.
High-resolution, high-penetration depth activatable probes are needed for in-vivo imaging of enzyme activity. In this
paper, we will describe the contrast mechanism of a new photoacoustic activatable probe that changes its excitation
lifetime upon activation. The excitation decay of methylene blue (MB), a chromophore commonly used in therapeutic
and diagnostic applications, is probed by photoacoustic lifetime contrast imaging (PLCI). The monomer of the dye
presents a high-quantum yield of intersystem-crossing and long lifetime (70 μs) whereas the dimer is statically quenched
with a short lifetime (a few ns). This forms the basis of a highly sensitive contrast mechanism between monomers and
dimers. Two dimerization models - one using sodium sulfate, the other using sodium dodecyl sulfate - were applied to
control the monomer-to-dimer ratio in MB solutions. Preliminary results show that the photoacoustic signal of a dimer
solution is efficiently suppressed (< 20 dB) due to their short lifetime compared to the monomer sample. Flash-photolysis
of the same solutions reveals a 99% decrease in transient absorption confirming PLCI results. This contrast
mechanism can be applied to design a MB dual-labeled activatable probe bound by an enzyme-specific cleavable peptide
linker. When the probe is cleaved by its target, MB molecules will separate by molecular diffusion and recover their long
excitation lifetime enabling their detection by PLCI. Our long-term goal is to investigate enzyme-specific imaging in
small animals and establish pre-clinical data for translational research and implementation of the technology in clinical
applications.
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