Leveraging ToF-SIMS imaging to investigate tenofovir disoproxil fumarate degradation at excipient interfaces in oral compressed tablets.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has been used to study the hydrolysis of tenofovir disoproxil fumarate (TDF) to tenofovir monosoproxil (TM) within an oral compressed tablet. The ToF-SIMS images displayed a heterogenous distribution of the matrix components. Evaluation of the TM distribution revealed that it was primarily co-localized with areas of higher excipient concentration pointing toward excipient driven degradation. To support these observations, a compatibility study of TDF with each tablet component was performed via liquid chromatography. The ToF-SIMS imaging and compatibility study indicated that the excipient, Avicel® PH-102, was the primary driver of TM formation in the tablet. The hydrolysis degradation mechanism within the tablet is further rationalized through discussion of chemical and physical properties of the matrix components. The sum of this work demonstrates a new analytical workflow for probing and understanding matrix driven degradation in oral compressed tablets utilizing ToF-SIMS imaging.

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.

A Photoacoustic-Fluorescent Imaging Probe for Proteolytic Gingipains Expressed by Porphyromonas gingivalis.

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU mL-1 bacteria in vitro. RgpB activity was imaged in porcine jaws with low-nanomolar sensitivity. Diagnostic efficacy was evaluated in gingival crevicular fluid samples from subjects with and without periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson's r=0.71). Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, with relevance and potential utility for disease models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.

High-resolution ultrasonography of gingival biomarkers for periodontal diagnosis in healthy and diseased subjects.

OBJECTIVE: To determine the capacity of ultrasonographic image-based measurements of gingival height and alveolar bone level for monitoring periodontal health and disease. METHODS: Sixteen subjects were recruited from patients scheduled to receive dental care and classified as periodontally healthy (n = 10) or diseased (n = 6) according to clinical guidelines. A 40-MHz ultrasound system was used to measure gingival recession, gingival height, alveolar bone level, and gingival thickness from 66 teeth for comparison to probing measurements of pocket depth and clinical attachment level. Interexaminer variability and comparison between ultrasound measurements and probing measurements was performed via Bland-Altman analysis. RESULTS: Gingival recession and its risk in non-recessed patients could be determined via measurement of the supra- and subgingival cementoenamel junction relative to the gingival margin. Interexaminer bias for ultrasound image analysis was negligible (<0.10 mm) for imaged gingival height (iGH) and 0.45 mm for imaged alveolar bone level (iABL). Diseased subjects had significantly higher imaging measurements (iGH, iABL) and clinical measurements (probing pocket depth, clinical attachment level) than healthy subjects (p < 0.05). Subtraction of the average biologic width from iGH resulted in 83% agreement (≤1 mm difference) between iGH and probing pocket depth measurements. CONCLUSIONS: Ultrasonography has an equivalent diagnostic capacity as gold-standard physical probing for periodontal metrics while offering more detailed anatomical information.

A Photoacoustic Contrast Agent for miR-21 via NIR Fluorescent Hybridization Chain Reaction.

Nucleic acids are well-established biomarkers of cancer with immense value in diagnostics and basic research. However, strategies to monitor these species in tissue can be challenging due to the need for amplification of imaging signal from low analyte concentrations with high specificity. Photoacoustic (PA) imaging is gaining traction for molecular imaging of proteins, small biomolecules, and nucleic acids by coupling pulsed near-infrared (NIR) excitation with broadband acoustic detection. This work introduces a PA nucleic acid contrast agent that harnesses NIR fluorophore and quencher-tagged hybridization chain reaction (HCR) for signal amplification. This HCR probe was designed to enable contact quenching between NIR dye-quencher pairs by coercing their direct alignment when miR-21, a microRNA cancer biomarker, is detected. The probe demonstrated a ratiometric PA limit of detection of 148 pM miR-21, sequence specificity against one- and two-base mutations, and selectivity over other microRNAs. It was further tested in live human ovarian cancer (SKOV3) and noncancerous (HEK 293T) cells to exemplify in situ PA activation based on differences in endogenous miR-21 regulation (p = 0.0002). The probe was lastly tested in tissue mimicking phantoms to exemplify sustained contrast in centimeter-range depths and 85.3% photostability after 15 min of laser irradiation. The probe's miR-21-specific activation and its ability to maintain contrast in biologically relevant absorbing and scattering media support its consideration for live-cell PA microscopy and potential cancer diagnostics. Results from this probe also underscore the combined detection power between ratiometric PA signaling and strand amplification for more sensitive DNA-based PA sensors.

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.

A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected Cells for Accurate Detection and Rapid Inhibitor Screening.

The main protease (Mpro ) and papain-like protease (PLpro ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 Mpro and PLpro . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

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.

Modulation of Gold Nanorod Growth via the Proteolysis of Dithiol Peptides for Enzymatic Biomarker Detection.

Gold nanorods possess optical properties that are tunable and highly sensitive to variations in their aspect ratio (length/width). Therefore, the development of a sensing platform where the gold nanorod morphology (i.e., aspect ratio) is modulated in response to an analyte holds promise in achieving ultralow detection limits. Here, we use a dithiol peptide as an enzyme substrate during nanorod growth. The sensing mechanism is enabled by the substrate design, where the dithiol peptide contains an enzyme cleavage site in-between cysteine amino acids. When cleaved, the peptide dramatically impacts gold nanorod growth and the resulting optical properties. We demonstrate that the optical response can be correlated with enzyme concentration and achieve a 45 pM limit of detection. Furthermore, we extend this sensing platform to colorimetrically detect tumor-associated inhibitors in a biologically relevant medium. Overall, these results present a subnanomolar method to detect proteases that are critical biomarkers found in cancers, infectious diseases, and inflammatory disorders.

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.

Photoacoustic Imaging as a Tool for Assessing Hair Follicular Organization.

Follicular unit extraction (FUE) and follicular unit transplantation (FUT) account for 99% of hair transplant procedures. In both cases, it is important for clinicians to characterize follicle density for treatment planning and evaluation. The existing gold-standard is photographic examination. However, this approach is insensitive to subdermal hair and cannot identify follicle orientation. Here, we introduce a fast and non-invasive imaging technique to measure follicle density and angles across regions of varying density. We first showed that hair is a significant source of photoacoustic signal. We then selected regions of low, medium, and high follicle density and showed that photoacoustic imaging can measure the density of follicles even when they are not visible by eye. We performed handheld imaging by sweeping the transducer across the imaging area to generate 3D images via maximum intensity projection. Background signal from the dermis was removed using a skin tracing method. Measurement of follicle density using photoacoustic imaging was highly correlated with photographic determination (R2 = 0.96). Finally, we measured subdermal follicular angles-a key parameter influencing transection rates in FUE.

Multispectral Nanoparticle Tracking Analysis for the Real-Time and Label-Free Characterization of Amyloid-ß Self-Assembly In Vitro.

The deposition of amyloid ß (Aß) plaques and fibrils in the brain parenchyma is a hallmark of Alzheimer's disease (AD), but a mechanistic understanding of the role Aß plays in AD has remained unclear. One important reason could be the limitations of current tools to size and count Aß fibrils in real time. Conventional techniques from molecular biology largely use ensemble averaging; some microscopy analyses have been reported but suffer from low throughput. Nanoparticle tracking analysis is an alternative approach developed in the past decade for sizing and counting particles according to their Brownian motion; however, it is limited in sensitivity to polydisperse solutions because it uses only one laser. More recently, multispectral nanoparticle tracking analysis (MNTA) was introduced to address this limitation; it uses three visible wavelengths to quantitate heterogeneous particle distributions. Here, we used MNTA as a label-free technique to characterize the in vitro kinetics of Aß1-42 aggregation by measuring the size distributions of aggregates during self-assembly. Our results show that this technology can monitor the aggregation of 106-108 particles/mL with a temporal resolution between 15 and 30 min. We corroborated this method with the fluorescent Thioflavin-T assay and transmission electron microscopy (TEM), showing good agreement between the techniques (Pearson's r = 0.821, P < 0.0001). We also used fluorescent gating to examine the effect of ThT on the aggregate size distribution. Finally, the biological relevance was demonstrated via fibril modulation in the presence of a polyphenolic Aß disruptor. In summary, this approach measures Aß assembly similar to ensemble-type measurements but with per-fibril resolution.

Noninvasive staging of pressure ulcers using photoacoustic imaging.

Ulcers including pressure ulcers and diabetic foot ulcers damage the skin and underlying tissue in people with compromised blood circulation. They are classified into four stages of severity and span from mild reddening of the skin to tissue damage and muscle/bone infections. Here, we used photoacoustic imaging as a noninvasive method for detecting early tissue damage that cannot be visually observed while also staging the disease using quantitative image analysis. We used a mouse model of pressure ulcers by implanting subdermal magnets in the dorsal flank and periodically applying an external magnet to the healed implant site. The magnet-induced pressure was applied in cycles, and the extent of ulceration was dictated by the number of cycles. We used both laser- and light-emitting diode (LED)-based photoacoustic imaging tools with 690 nm excitation to evaluate the change in photoacoustic signal and depth of injury. Using laser-based photoacoustic imaging system, we found a 4.4-fold increase in the photoacoustic intensity in stage I vs. baseline (no pressure). We also evaluated the depth of injury using photoacoustics. We measured a photoacoustic ulcer depth of 0.38 ± 0.09 mm, 0.74 ± 0.11 mm, 1.63 ± 0.4 mm, and 2.7 ± 0.31 mm (n = 4) for stages I-IV, respectively. The photoacoustic depth differences between each stage were significant (p < 0.05). We also used an LED-based photoacoustic imaging system to detect early stage (stage I) pressure ulcers and observed a 2.5-fold increase in photoacoustic signal. Importantly, we confirmed the capacity of this technique to detect dysregulated skin even before stage I ulcers have erupted. We also observed significant changes in photoacoustic intensity during healing suggesting that this approach can monitor therapy. These findings were confirmed with histology. These results suggest that this photoacoustic-based approach might have clinical value for monitoring skin diseases including pressure ulcers.

Listening for the therapeutic window: Advances in drug delivery utilizing photoacoustic imaging.

The preclinical landscape of photoacoustic imaging has experienced tremendous growth in the past decade. This non-invasive imaging modality augments the spatiotemporal capabilities of ultrasound with optical contrast. While it has principally been investigated for diagnostic applications, many recent reports have described theranostic delivery systems and drug monitoring strategies using photoacoustics. Here, we provide an overview of the progress to date while highlighting work in three specific areas: theranostic nanoparticles, real-time drug monitoring, and stem cell ("living drug") tracking. Additionally, we discuss the challenges that remain to be addressed in this burgeoning field.

Strategies for Image-Guided Therapy, Surgery, and Drug Delivery Using Photoacoustic Imaging.

Photoacoustic imaging is a rapidly maturing imaging modality in biological research and medicine. This modality uses the photoacoustic effect ("light in, sound out") to combine the contrast and specificity of optical imaging with the high temporal resolution of ultrasound. The primary goal of image-guided therapy, and theranostics in general, is to transition from conventional medicine to precision strategies that combine diagnosis with therapy. Photoacoustic imaging is well-suited for noninvasive guidance of many therapies and applications currently being pursued in three broad areas. These include the image-guided resection of diseased tissue, monitoring of disease states, and drug delivery. In this review, we examine the progress and strategies for development of photoacoustics in these three key areas with an emphasis on the value photoacoustics has for image-guided therapy.

Etiology and Measurement of Peri-Implant Crestal Bone Loss (CBL).

The etiology of peri-implant crestal bone loss is today better understood and certain factors proposed in the past have turned out to not be of concern. Regardless, the incidence of crestal bone loss remains higher than necessary and this paper reviews current theory on the etiology with a special emphasis on traditional and innovative methods to assess the level of crestal bone around dental implants that will enable greater sensitivity and specificity and significantly reduce variability in bone loss measurement.

Photoacoustic imaging for monitoring periodontal health: A first human study.

The gold-standard periodontal probe is an aging tool that can detect periodontitis and monitor gingival health but is highly error-prone, does not fully characterize the periodontal pocket, and causes pain. Photoacoustic imaging is a noninvasive technique that can address these limitations. Here, a range of ultrasound frequencies between 16-40 MHz were used to image the periodontium and a contrast medium based on cuttlefish ink was used to label the pockets. A 40 MHz ultrasound frequency could spatially resolve the periodontal anatomy, including tooth, gum, gingival margin, and gingival thickness of tooth numbers 7-10 and 22-27. The photoacoustic-ultrasound measurements were more precise (0.01 mm) than those taken with physical probes by a dental hygienist. Furthermore, the full geometry of the pockets could be visualized with relative standard deviations of 10% (n = 5). This study shows the potential for non-invasive monitoring of periodontal health with photoacoustic-ultrasound imaging in the dental clinic.