Supramolecular Loading of DNA Hydrogels with Dye-Drug Conjugates for Real-Time Photoacoustic Monitoring of Chemotherapy.

A longstanding problem with conventional cancer therapy is the nonspecific distribution of chemotherapeutics. Monitoring drug release in vivo via noninvasive bioimaging can thus have value, but it is difficult to distinguish loaded from released drug in live tissue. Here, this work describes an injectable supramolecular hydrogel that allows slow and trackable release of doxorubicin (Dox) via photoacoustic (PA) tomography. Dox is covalently linked with photoacoustic methylene blue (MB) to monitor Dox before, during, and after release from the hydrogel carrier. The conjugate (MB-Dox) possesses an IC50 of 161.4 × 10-9  m against human ovarian carcinoma (SKOV3) cells and loads into a DNA-clad hydrogel with 91.3% loading efficiency due to MB-Dox's inherent intramolecular affinity to DNA. The hydrogel is biodegradable by nuclease digestion, which causes gradual release of MB-Dox. This release rate is tunable based on the wt% of the hydrogel. This hydrogel maintains distinct PA contrast on the order of days when injected in vivo and demonstrates activatable PA spectral shifts   during hydrogel degradation. The released and loaded payload can be imaged relative to live tissue via PA and ultrasound signal being overlaid in real-time. The hydrogel slowed the rate of the murine intraperitoneal tumor growth 72.2% more than free Dox.

Di-Arginine Additives for Dissociation of Gold Nanoparticle Aggregates: A Matrix-Insensitive Approach with Applications in Protease Detection.

We report the reversible aggregation of gold nanoparticles (AuNPs) assemblies via a di-arginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that the HS-PEG thiol group has a higher affinity for the AuNP surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies' dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNP suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates, or even seawater. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favored. Moreover, starting with AuNPs already aggregated ensured the absence of a background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNP assemblies and solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluids. Trypsin was chosen as the model enzyme, and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrates. The increase of positive charge of the HS-PEG-peptide conjugate quenched the dissociation capacity of the HS-PEG molecules, which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine.

Photoacoustic Enhancement of Ferricyanide-Treated Silver Chalcogenide-Coated Gold Nanorods.

Plasmonic gold nanorods (AuNRs) are often employed as photoacoustic (PA) contrast agents due to their ease of synthesis, functionalization, and biocompatibility. These materials can produce activatable signals in response to a change in optical absorbance intensity or absorbance wavelength. Here, we report a surprising finding: Ag2S/Se-coated AuNRs have a ~40-fold PA enhancement upon addition of an oxidant but with no change in absorption spectra. We then study the mechanism underlying this enhancement. Electron micrographs and absorption spectra show good colloidal stability and retention of the core-shell structure after potassium hexacyanoferrate(III) (HCF) addition, ruling out aggregation and morphology-induced PA enhancement. X-ray diffraction data showed no changes, ruling out crystallographic phase changes upon HCF addition, thus leading to induced PA enhancement. Attenuated total reflectance-Fourier transform infrared spectroscopy and zeta potential analysis suggest that PA enhancement is driven by the irreversible displacement of hexadecyltrimethylammonium bromide with HCF. This is further confirmed using elemental mapping with energy-dispersive X-ray analysis. PA characterization after HCF addition showed a four-fold increase in the Grüneisen parameter (Γ), thus resulting in PA enhancement. The PA enhancement is not seen in uncoated AuNRs or spherical particles. Two possible mechanisms for PA enhancement are proposed: first, the photo-induced redox heating at the Ag2S/Se shell-HCF interface, resulting in an increase in temperature-dependent Γ, and second, an enhanced electrostriction response due to HCF adsorption on a layered plasmonic nanoparticle surface, resulting in a high thermal expansion coefficient (ß) that is directly proportional to Γ.

Photoacoustic imaging phantoms for assessment of object detectability and boundary buildup artifacts.

Standardized phantoms and test methods are needed to accelerate clinical translation of emerging photoacoustic imaging (PAI) devices. Evaluating object detectability in PAI is challenging due to variations in target morphology and artifacts including boundary buildup. Here we introduce breast fat and parenchyma tissue-mimicking materials based on emulsions of silicone oil and ethylene glycol in polyacrylamide hydrogel. 3D-printed molds were used to fabricate solid target inclusions that produced more filled-in appearance than traditional PAI phantoms. Phantoms were used to assess understudied image quality characteristics (IQCs) of three PAI systems. Object detectability was characterized vs. target diameter, absorption coefficient, and depth. Boundary buildup was quantified by target core/boundary ratio, which was higher in transducers with lower center frequency. Target diameter measurement accuracy was also size-dependent and improved with increasing transducer frequency. These phantoms enable evaluation of multiple key IQCs and may support development of comprehensive standardized test methods for PAI devices.

Peptide-Induced Fractal Assembly of Silver Nanoparticles for Visual Detection of Disease Biomarkers.

We report the peptide-programmed fractal assembly of silver nanoparticles (AgNPs) in a diffusion-limited aggregation (DLA) mode, and this change in morphology generates a significant color change. We show that peptides with specific repetitions of defined amino acids (i.e., arginine, histidine, or phenylalanine) can induce assembly and coalescence of the AgNPs (20 nm) into a hyperbranched structure (AgFSs) (∼2 µm). The dynamic process of this assembly was systematically investigated, and the extinction of the nanostructures can be modulated from 400 to 600 nm by varying the peptide sequences and molar ratio. According to this rationale, two strategies of SARS-CoV-2 detection were investigated. The activity of the main protease (Mpro) involved in SARS-CoV-2 was validated with a peptide substrate that can bridge the AgNPs after the proteolytic cleavage. A sub-nanomolar limit of detection (0.5 nM) and the capacity to distinguish by the naked eye in a wide concentration range (1.25-30 nM) were achieved. Next, a multichannel sensor-array based on multiplex peptides that can visually distinguish SARS-CoV-2 proteases from influenza proteases in doped human samples was investigated.

Impact of skin tone on photoacoustic oximetry and tools to minimize bias.

The major optical absorbers in tissue are melanin and oxy/deoxy-hemoglobin, but the impact of skin tone and pigmentation on biomedical optics is still not completely understood or adequately addressed. Melanin largely governs skin tone with higher melanin concentration in subjects with darker skin tones. Recently, there has been extensive debate on the bias of pulse oximeters when used with darker subjects. Photoacoustic (PA) imaging can measure oxygen saturation similarly as pulse oximeters and could have value in studying this bias. More importantly, it can deconvolute the signal from the skin and underlying tissue. Here, we studied the impact of skin tone on PA signal generation, depth penetration, and oximetry. Our results show that subjects with darker skin tones exhibit significantly higher PA signal at the skin surface, reduced penetration depth, and lower oxygen saturation compared to subjects with lighter skin tones. We then suggest a simple way to compensate for these signal differences.

Monitoring peripheral hemodynamic response to changes in blood pressure via photoacoustic imaging.

Chronic wounds and amputations are common in chronic kidney disease patients needing hemodialysis (HD). HD is often complicated by drops in blood pressure (BP) called intra-dialytic hypotension. Whether intra-dialytic hypotension is associated with detectable changes in foot perfusion, a risk factor for wound formation and impaired healing remains unknown. Photoacoustic (PA) imaging is ideally suited to study perfusion changes. We scanned the feet of 20 HD and 11 healthy subjects. HD patients were scanned before and after a dialysis session whereas healthy subjects were scanned twice at rest and once after a 10 min exercise period while BP was elevated. Healthy (r = 0.70, p < 0.0001) and HD subjects (r = 0.43, p < 0.01) showed a significant correlation between PA intensity and systolic BP. Furthermore, HD cohort showed a significantly reduced PA response to changes in BP compared to the healthy controls (p < 0.0001), showing that PA can monitor hemodynamic changes due to changes in BP.

Photoacoustic monitoring of angiogenesis predicts response to therapy in healing wounds.

Chronic wounds are a major health problem that cause the medical infrastructure billions of dollars every year. Chronic wounds are often difficult to heal and cause significant discomfort. Although wound specialists have numerous therapeutic modalities at their disposal, tools that could three dimensional-map wound bed physiology and guide therapy do not exist. Visual cues are the current standard but are limited to surface assessment; clinicians rely on experience to predict response to therapy. Photoacoustic (PA) ultrasound (US) is a non-invasive, hybrid imaging modality that can solve these major limitations. PA relies on the contrast generated by haemoglobin in blood which allows it to map local angiogenesis, tissue perfusion and oxygen saturation-all critical parameters for wound healing. This work evaluates the use of PA-US to monitor angiogenesis and stratify patients responding versus not-responding to therapy. We imaged 19 patients with 22 wounds once a week for at least 3 weeks. Our findings suggest that PA imaging directly visualises angiogenesis. Patients responding to therapy showed clear signs of angiogenesis and an increased rate of PA increase (p = 0.002). These responders had a significant and negative correlation between PA intensity and wound size. Hypertension was correlated to impaired angiogenesis in non-responsive patients. The rate of PA increase and hence the rate of angiogenesis was able to predict healing times within 30 days from the start of monitoring (power = 88%, alpha = 0.05). This early response detection system could help inform management and treatment strategies while improving outcomes and reducing costs.

Ultrasmall gold nanorod-polydopamine hybrids for enhanced photoacoustic imaging and photothermal therapy in second near-infrared window.

Gold nanorods (GNRs) have attracted great interest for photo-mediated biomedicines due to their tunable and high optical absorption, high photothermal conversion efficiency and facile surface modifiability. GNRs that have efficient absorption in second near-infrared (NIR-II) window hold further promise in bio-applications due to low background signal from tissue and deep tissue penetration. However, bare GNRs readily undergo shape deformation (termed as 'melting effect') during the laser illumination losing their unique localized surface plasmon resonance (LSPR) properties, which subsequently leads to PA signal attenuation and decreased photothermal efficiency. Polydopamine (PDA) is a robust synthetic melanin that has broad absorption and high photothermal conversion. Herein, we coated GNRs with PDA to prepare photothermally robust GNR@PDA hybrids for enhanced photo-mediated theranostic agents. Ultrasmall GNRs (SGNRs) and conventional large GNRs (LGNRs) that possess similar LSPR characteristics as well as GNR@PDA hybrids were compared side-by-side in terms of the size-dependent photoacoustic (PA) imaging, photothermal therapy (PTT), and structural stability. In vitro experiments further demonstrated that SGNR@PDA showed 95% ablation of SKOV3 ovarian cancer cells, which is significantly higher than that of LGNRs (66%) and SGNRs (74%). Collectively, our PDA coating strategy represents a rational design for enhanced PA imaging and efficient PTT via a nanoparticle, i.e., nanotheranostics.

A Charge-Switchable Zwitterionic Peptide for Rapid Detection of SARS-CoV-2 Main Protease.

The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (Mpro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect Mpro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by Mpro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t<10 min). We determine a limit of detection for Mpro in breath condensate matrices <10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.

Peptidic Sulfhydryl for Interfacing Nanocrystals and Subsequent Sensing of SARS-CoV-2 Protease.

There is a need for surveillance of COVID-19 to identify individuals infected with SARS-CoV-2 coronavirus. Although specific, nucleic acid testing has limitations in terms of point-of-care testing. One potential alternative is the nonstructural protease (nsp5, also known as Mpro/3CLpro) implicated in SARS-CoV-2 viral replication but not incorporated into virions. Here, we report a divalent substrate with a novel design, (Cys)2-(AA)x-(Asp)3, to interface gold colloids in the specific presence of Mpro leading to a rapid and colorimetric readout. Citrate- and tris(2-carboxyethyl)phosphine (TCEP)-AuNPs were identified as the best reporter out of the 17 ligated nanoparticles. Furthermore, we empirically determined the effects of varying cysteine valence and biological media on the sensor specificity and sensitivity. The divalent peptide was specific to Mpro, that is, there was no response when tested with other proteins or enzymes. Furthermore, the Mpro detection limits in Tris buffer and exhaled breath matrices are 12.2 and 18.9 nM, respectively, which are comparable to other reported methods (i.e., at low nanomolar concentrations) yet with a rapid and visual readout. These results from our work would provide informative rationales to design a practical and noninvasive alternative for COVID-19 diagnostic testing-the presence of viral proteases in biofluids is validated.

Photoacoustic imaging to monitor outcomes during hyperbaric oxygen therapy: validation in a small cohort and case study in a bilateral chronic ischemic wound.

Hyperbaric oxygen therapy (HBO2) is a common therapeutic modality that drives oxygen into hypoxic tissue to promote healing. Here, ten patients undergoing HBO2 underwent PA oximetry of the left radial artery and forearm pre- and post-HBO2; this cohort validated the use of PA imaging in HBO2. There was a significant increase in radial artery oxygenation after HBO2 (p = 0.002) in the validation cohort. We also include a case study: a non-diabetic male in his 50s (HB 010) presenting with bilateral ischemic and gangrenous wounds. HB 010 showed higher perfusion and oxygen saturation on the right foot than the left after HBO2 which correlated with independent surgical observations. Imaging assisted with limb salvage treatment. Hence, this work shows that PA imaging can measure changes in arterial oxygen saturation due to HBO2; it can also produce 3D maps of tissue oxygenation and evaluate response to therapy during HBO2.

A fiber optic photoacoustic sensor for real-time heparin monitoring.

Heparin is a common anticoagulant, but heparin overdose is a common intensive care unit (ICU) medication error due to the narrow therapeutic window of heparin. Conventional methods to monitoring heparin suffer from long turnaround time, the need for skilled personnel, and low frequency of sampling. To overcome these issues, we describe here a fiber optic photoacoustic (PA) sensor for real-time heparin monitoring. The proposed sensor was validated with in vitro testing and in a simulated in vivo model using the following samples: (1) phosphate-buffered saline (PBS), (2) spiked human plasma, (3) spiked whole human blood, and (4) clinical samples from patients treated with heparin. Samples were validated by comparing the PA signal to the activated partial thromboplastin time (aPTT) as well as the activated clotting time (ACT). Importantly, the proposed sensor has a short turnaround time (3 min) and a limit of detection of 0.18 U/ml in whole human blood. The PA signal is linear with heparin dose and correlates with the aPTT value (Pearson's r = 0.99). The PA signal from 32 clinical samples collected from eight patients linearly correlated with ACT values (Pearson's r = 0.89, in vitro; Pearson's r = 0.93, simulated in vivo). The PA signal was also validated against the cumulative heparin dose (Pearson's r = 0.94, in vitro; Pearson's r = 0.96, simulated in vivo). This approach could have applications in both in vitro and real-time in vivo heparin monitoring.

Versatile Polymer Nanocapsules via Redox Competition.

Polymer nanocapsules have demonstrated significant value in materials science and biomedical technology, but require complicated and time-consuming synthetic steps. We report here the facile synthesis of monodisperse polymer nanocapsules via a redox-mediated kinetic strategy from two simple molecules: dopamine and benzene-1,4-dithiol (BDT). Specifically, BDT forms core templates and modulates the oxidation kinetics of dopamine into polydopamine (PDA) shells. These uniform nanoparticles can be tuned between ≈70 and 200 nm because the core diameter directly depends on BDT while the shell thickness depends on dopamine. The supramolecular core can then rapidly disassemble in organic solvents to produce PDA nanocapsules. Such nanocapsules exhibit enhanced physicochemical performance (e.g., loading capacity, photothermal transduction, and anti-oxidation) versus their solid counterparts. Particularly, this method enables a straightforward encapsulation of functional nanoparticles providing opportunities for designing complex nanostructures such as yolk-shell nanoparticles.

Point-of-Care Ultrasound as a Tool to Assess Wound Size and Tissue Regeneration after Skin Grafting.

Chronic wounds can be difficult to heal and are often accompanied by pain and discomfort. Multiple skin substitutes or cellularized/tissue-based skin products have been used in an attempt to facilitate closure of complex wounds. Allografts from cadaveric sources have been a viable option in achieving such closure. However, early assessment of graft incorporation has been difficult clinically, often with delayed evidence of failure. Visual cues to assess graft integrity have been limited and remain largely superficial at the skin surface. Furthermore, currently used optical imaging techniques can penetrate only a few millimeters deep into tissue. Ultrasound (US) imaging offers a potential solution to address this limitation. This work evaluates the use of US to monitor wound healing and allograft integration. We used a commercially available dual-mode (US and photoacoustic) scanner operating only in US mode. We compared the reported wound size from the clinic with the size measured using US in 45 patients. Two patients from this cohort received an allogenic skin graft and underwent multiple US scans over a 110-d period. All data were processed by two independent analysts; one of them was blinded to the study. We measured change in US intensity and wound contraction as a function of time. Our results revealed a strong correlation (R2 = 0.81, p < 0.0001) between clinically and US-measured wound sizes. Wound contraction >91% was seen in both patients after skin grafting. An inverse relationship between wound size and US intensity (R2 = 0.77, p < 0 .0001) indicated that the echogenicity of the wound bed increases as healthy cells infiltrate the allograft matrix, regenerating and leading to healthy tissue and re-epithelization. This work indicates that US can be used to measure wound size and visualize tissue regeneration during the healing process.

Mapping Aerosolized Saliva on Face Coverings for Biosensing Applications.

Facemasks in congregate settings prevent the transmission of SARS-CoV-2 and help control the ongoing COVID-19 global pandemic because face coverings can arrest transmission of respiratory droplets. While many groups have studied face coverings as personal protective equipment, these respiratory droplets can also serve as a diagnostic fluid to report on health state; surprisingly, studies of face coverings from this perspective are quite limited. Here, we determined the concentration and distribution of aerosolized saliva (via α-amylase levels) captured on various face coverings. Our results showed that α-amylase accumulated on face coverings in a time-dependent way albeit at different levels, e.g., neck gaiters and surgical masks captured about 3-fold more α-amylase than cloth masks and N95 respirators. In addition, the saliva aerosols were primarily detected on the inner layer of multilayered face coverings. We also found that the distribution of salivary droplets on the mask correlated with the morphologies of face coverings as well as their coherence to the face curvature. These findings motivated us to extend this work and build multifunctional sensing strips capable of detecting biomarkers in situ to create "smart" masks. The work highlights that face coverings are promising platforms for biofluid collection and colorimetric biosensing, which bode well for developing surveillance tools for airborne diseases.

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.

Motion-compensated noninvasive periodontal health monitoring using handheld and motor-based photoacoustic-ultrasound imaging systems.

Simultaneous visualization of the teeth and periodontium is of significant clinical interest for image-based monitoring of periodontal health. We recently reported the application of a dual-modality photoacoustic-ultrasound (PA-US) imaging system for resolving periodontal anatomy and periodontal pocket depths in humans. This work utilized a linear array transducer attached to a stepper motor to generate 3D images via maximum intensity projection. This prior work also used a medical head immobilizer to reduce artifacts during volume rendering caused by motion from the subject (e.g., breathing, minor head movements). However, this solution does not completely eliminate motion artifacts while also complicating the imaging procedure and causing patient discomfort. To address this issue, we report the implementation of an image registration technique to correctly align B-mode PA-US images and generate artifact-free 2D cross-sections. Application of the deshaking technique to PA phantoms revealed 80% similarity to the ground truth when shaking was intentionally applied during stepper motor scans. Images from handheld sweeps could also be deshaken using an LED PA-US scanner. In ex vivo porcine mandibles, pigmentation of the enamel was well-estimated within 0.1 mm error. The pocket depth measured in a healthy human subject was also in good agreement with our prior study. This report demonstrates that a modality-independent registration technique can be applied to clinically relevant PA-US scans of the periodontium to reduce operator burden of skill and subject discomfort while showing potential for handheld clinical periodontal imaging.

Gold Nanorod-Melanin Hybrids for Enhanced and Prolonged Photoacoustic Imaging in the Near-Infrared-II Window.

Photoacoustic (PA) imaging holds great promise as a noninvasive imaging modality. Gold nanorods (GNRs) with absorption in the second near-infrared (NIR-II) window have emerged as excellent PA probes because of their tunable optical absorption, surface modifiability, and low toxicity. However, pristine GNRs often undergo shape transition upon laser illumination due to thermodynamic instability, leading to a reduced PA signal after a few seconds of imaging. Here, we report monodisperse GNR-melanin nanohybrids where a tunable polydopamine (PDA) coating was conformally coated on GNRs. GNR@PDAs showed a threefold higher PA signal than pristine GNRs due to the increased optical absorption, cross-sectional area, and thermal confinement. More importantly, the PA signal of GNR@PDAs only decreased by 29% during the 5 min of laser illumination in the NIR-II window, while significant attenuation (77%) was observed for GNRs. The GNR@PDAs maintained 87% of its original PA signal in vivo even after 10 min of laser illumination. This PDA-enabled strategy affords a rational design for robust PA imaging probes and provides more opportunities for other types of photomediated biomedicines, such as photothermal and photodynamic regimens.