Determining topical product bioequivalence with stimulated Raman scattering microscopy.

Generic drugs are essential for affordable medicine and improving accessibility to treatments. Bioequivalence (BE) is typically demonstrated by assessing a generic product's pharmacokinetics (PK) relative to a reference-listed drug (RLD). Accurately estimating cutaneous PK (cPK) at or near the site of action can be challenging for locally acting topical products. Certain cPK approaches are available for assessing local bioavailability (BA) in the skin. Stimulated Raman scattering (SRS) microscopy has unique capabilities enabling continuous, high spatial and temporal resolution and quantitative imaging of drugs within the skin. In this paper, we developed an approach based on SRS and a polymer-based standard reference for the evaluation of topical product BA and BE in human skin ex vivo. BE assessment of tazarotene-containing formulations was achieved using cPK parameters obtained within different skin microstructures. The establishment of BE between the RLD and an approved generic product was successfully demonstrated. Interestingly, within the constraints of the current study design the results suggest similar BA between the tested gel formulation and the reference cream formulation, despite the differences in the formulation/dosage form. Another formulation containing polyethylene glycol as the vehicle was demonstrated to be not bioequivalent to the RLD. Compared to using the SRS approach without a standard reference, the developed approach enabled more consistent and reproducible results, which is crucial in BE assessment. The abundant information from the developed approach can help to systematically identify key areas of study design that will enable a better comparison of topical products and support an assessment of BE.

Quantitative analysis of drug tablet aging by fast hyper-spectral stimulated Raman scattering microscopy.

Pharmaceutical development of solid-state formulations requires testing active pharmaceutical ingredients (API) and excipients for uniformity and stability. Solid-state properties such as component distribution and grain size are crucial factors that influence the dissolution profile, which greatly affect drug efficacy and toxicity, and can only be analyzed spatially by chemical imaging (CI) techniques. Current CI techniques such as near infrared microscopy and confocal Raman spectroscopy are capable of high chemical and spatial resolution but cannot achieve the measurement speeds necessary for integration into the pharmaceutical production and quality assurance processes. To fill this gap, we demonstrate fast chemical imaging by epi-detected sparse spectral sampling stimulated Raman scattering to quantify API and excipient degradation and distribution.

Development and in-vivo validation of a portable phosphorescence lifetime-based fiber-optic oxygen sensor.

Oxygenation is a crucial indicator of tissue viability and function. Oxygen tension ([Formula: see text]), i.e. the amount of molecular oxygen present in the tissue is a direct result of supply (perfusion) and consumption. Thus, measurement of [Formula: see text] is an effective method to monitor tissue viability. However, tissue oximetry sensors commonly used in clinical practice instead rely on measuring oxygen saturation ([Formula: see text]), largely due to the lack of reliable, affordable [Formula: see text] sensing solutions. To address this issue we present a proof-of-concept design and validation of a low-cost, lifetime-based oxygen sensing fiber. The sensor consists of readily-available off-the shelf components such as a microcontroller, a light-emitting diode (LED), an avalanche photodiode (APD), a temperature sensor, as well as a bright in-house developed porphyrin molecule. The device was calibrated using a benchtop setup and evaluated in three in vivo animal models. Our findings show that the new device design in combination with the bright porphyrin has the potential to be a useful and accurate tool for measuring [Formula: see text] in tissue, while also highlighting some of the limitations and challenges of oxygen measurements in this context.

Calculation of Tissue Oxygenation via an Inverse Boundary Problem for Transcutaneous Oxygenation Wearable Applications.

In this article, we present a toolset to fully leverage a previously developed transcutaneous oxygenation monitor (TCOM) wearable technology to accurately measure skin oxygenation values. We describe numerical models and experimental characterization techniques that allow for the extraction of precise tissue oxygenation measurements. The numerical model is based on an inverse boundary problem of the parabolic equation with Dirichlet boundary conditions. To validate this model and characterize the diffusion of oxygen through the oxygen sensing materials, we designed a series of control/calibration experiments modeled after the device's clinical application using oxygenation values in the physiological range expected for healthy tissue. Our results demonstrate that it is possible to obtain accurate tissue pO2 measurements without the need for long equilibration times with a small wearable device.

Quantifying Inflammatory Response and Drug-Aided Resolution in an Atopic Dermatitis Model with Deep Learning.

Noninvasive quantification of dermal diseases aids efficacy studies and paves the way for broader enrollment in clinical studies across varied demographics. Related to atopic dermatitis, accurate quantification of the onset and resolution of inflammatory flare ups in the skin remains challenging because the commonly used macroscale cues do not necessarily represent the underlying inflammation at the cellular level. Although atopic dermatitis affects over 10% of Americans, the genetic underpinnings and cellular-level phenomena causing the physical manifestation of the disease require more clarity. Current gold standards of quantification are often invasive, requiring biopsies followed by laboratory analysis. This represents a gap in our ability to diagnose and study skin inflammatory disease as well as develop improved topical therapeutic treatments. This need can be addressed through noninvasive imaging methods and the use of modern quantitative approaches to streamline the generation of relevant insights. This work reports the noninvasive image-based quantification of inflammation in an atopic dermatitis mouse model on the basis of cellular-level deep learning analysis of coherent anti-Stokes Raman scattering and stimulated Raman scattering imaging. This quantification method allows for timepoint-specific disease scores using morphological and physiological measurements. The outcomes we show set the stage for applying this workflow to future clinical studies.

Cryoneurolysis with Injectable Ice Slurry Modulates Mechanical Skin Pain.

Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administration‒approved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.

Fast-acting and injectable cryoneurolysis device.

Cryoneurolysis is an opioid-sparing therapy for long-lasting and reversible reduction of pain. We developed a nerve-selective method for cryoneurolysis by local injection of ice-slurry (- 5 to - 6 °C) that induced decrease in nocifensive response starting from about a week after treatment and lasting up to 8 weeks. In this study, we test the hypothesis that injection of colder slurry leads to faster onset of analgesia. Colder slurry (- 9ºC) was injected around the rat sciatic nerve to induce cryoneurolysis. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on surrounding tissues. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths. Functional tests were used to assess changes in sensory and motor function in the treated hind paw. No inflammation or scarring was detected in surrounding skin and muscle tissues at day 7 post slurry injection. Functional tests showed rapid onset reduction in mechanical pain sensitivity starting from day 1 and lasting up to day 98. CARS imaging demonstrated disintegration of myelin sheaths post treatment followed by complete recovery of nerve structure by day 140. In this study we showed that colder slurry (- 9 °C) produces more rapid onset and longer duration of analgesia, while remaining nerve-selective.

Phosphorescent Microneedle Array for the Measurement of Oxygen Partial Pressure in Tissue.

The knowledge of the exact oxygen partial pressure in tissue is crucial for patient care and in the treatment of ischemic medical conditions. However, current methods to assess oxygen partial pressure in tissue suffer from a variety of disadvantages, including complex equipment and procedures that necessitate trained personnel. Additionally, the barrier function of the stratum corneum reduces oxygen exchange and can consequently hamper surface measurements of rapidly changing oxygen partial pressure in tissue. To overcome these challenges, a novel, easy-to-use technique to monitor the oxygen partial pressure in tissue using microneedle arrays (MNAs) has been developed. The MNAs can be made from poly(ethyl methacrylate) and poly(propyl methacrylate) and overcome the skin's barrier function to measure oxygen in the capillary bed and interstitial fluid of the skin. The MNAs' tips are embedded with an oxygen-sensitive phosphorescent metalloporphyrin, where the oxygen partial pressure inversely correlates to changes in both emission intensity and phosphorescence lifetime of the in-house developed red emitting Pt-core porphyrin. It was demonstrated that the oxygen-sensing MNAs are sufficiently robust to puncture human skin via rupture of the stratum corneum, and that the MNAs can detect changes in oxygen partial pressure in skin within the physiologically relevant range (0-160 mmHg). Additionally, the MNAs can be combined with a wearable wireless optical readout system, making these oxygen-sensing MNAs a novel wearable and portable method for user-friendly monitoring of oxygen partial pressure in skin.

Full Recovery after Multiple Treatments with Injectable Ice Slurry.

Background: Cryoneurolysis uses tissue cooling as an opioid-sparing, long-lasting treatment for peripheral nerve pain. A nerve-selective method for cryoneurolysis by local injection of ice-slurry was developed to allow cryoneurolysis to be performed with a standard needle and syringe, similar to peripheral nerve blocks. Since the treatment of patients with chronic pain may require repeated injections, we investigated the safety and tolerance of repeated treatments in a rat model. Methods: Three repeated ice-slurry treatments, given 6 weeks apart were performed around the rat sciatic nerve. Nerve and surrounding tissues were collected up to 4 months after the third treatment for analysis. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths and axon structure. Immunofluorescence (IF) staining was used to study effects on axon density. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on sciatic nerve and surrounding tissue. Results: Histologic and CARS image analysis of nerve tissue collected months after three injections demonstrated recovery of nerve structure, myelin organization and axon density to baseline levels, without any residual inflammation, scarring or neuroma formation. No inflammation or scarring was detected in surrounding skin and muscle tissues. Conclusion: Repeated ice-slurry injections cause temporary, nerve-selective and reversible changes in the peripheral nerve. There was no histologic damage to surrounding skin and muscle tissues. Repeated treatments with injectable ice-slurry for cryoneurolysis appear to be safe and well tolerated. Clinical studies for patients with chronic pain are warranted.

A Patient-Ready Wearable Transcutaneous CO2 Sensor.

Continuously monitoring transcutaneous CO2 partial pressure is of crucial importance in the diagnosis and treatment of respiratory and cardiac diseases. Despite significant progress in the development of CO2 sensors, their implementation as portable or wearable devices for real-time monitoring remains under-explored. Here, we report on the creation of a wearable prototype device for transcutaneous CO2 monitoring based on quantifying the fluorescence of a highly breathable CO2-sensing film. The developed materials are based on a fluorescent pH indicator (8-hydroxy-1,3,6-pyrenetrisulfonic acid trisodium salt or HPTS) embedded into hydrophobic polymer matrices. The film's fluorescence is highly sensitive to changes in CO2 partial pressure in the physiological range, as well as photostable and insensitive to humidity. The device and medical-grade films are based on our prior work on transcutaneous oxygen-sensing technology, which has been extensively validated clinically.

Quantitative Luminescence Photography of a Swellable Hydrogel Dressing with a Traffic-Light Response to Oxygen.

Sensor-integrated wound dressings are emerging tools applicable to a wide variety of medical applications from emergency triage to at-home monitoring. Uncomfortable, unnecessary wound dressing changes may be avoided by providing quantitative insight into tissue characteristics related to wound healing such as tissue oxygenation, pH, and exudate/transudate volume. Here, a simple cost-effective methodology for quantifying oxygen and pH in a swellable hydrogel dressing using a single photograph is presented. The red and green luminescence of a novel dendritic polyamine Pt-porphyrin and fluorescein conjugate quantitatively responds to oxygen and pH, respectively, and enables robust sensing. The porphyrin conjugate, when combined with a four-arm star polyethylene glycol (PEG) amine polymer, rapidly crosslinks at room temperature with an N-hydroxysuccinimide (NHS)-PEG crosslinker to form a color-changing hydrogel dressing with tunable swelling capabilities applicable to a variety of wound environments. An inexpensive digital single-lens reflex (DSLR) camera modified with bandpass filters captures the hydrogel luminescence using simple macroscopic photography, and conversion to HSB colorspace allows for intensity-independent image analysis of the hydrogels' dual modality response. The hydrogel formulation exhibits a robust and validated visible red-orange-green "traffic light" spectrum in response to oxygen changes, regardless of swelling state, pH, or autofluorescence from skin, thereby enabling the clinician friendly naked-eye feedback.

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging.

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development scientists to mechanistically understand topical bioavailability (BA). Semi-invasive techniques, such as tape-stripping, dermal microdialysis, or dermal open-flow microperfusion, all quantify macroscale cPK. While these techniques have provided vast cPK knowledge, the community lacks a mechanistic understanding of active pharmaceutical ingredient (API) penetration and permeation at the cellular level. One noninvasive approach to address microscale cPK is coherent Raman scattering imaging (CRI), which selectively targets intrinsic molecular vibrations without the need for extrinsic labels or chemical modification. CRI has two main methods-coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS)-that enable sensitive and selective quantification of APIs or inactive ingredients. CARS is typically utilized to derive structural skin information or visualize chemical contrast. In contrast, the SRS signal, which is linear with molecular concentration, is used to quantify APIs or inactive ingredients within skin stratifications. Although mouse tissue has commonly been utilized for cPK with CRI, topical BA and bioequivalence (BE) must ultimately be assessed in human tissue before regulatory approval. This paper presents a methodology to prepare and image ex vivo skin to be used in quantitative pharmacokinetic CRI studies in the evaluation of topical BA and BE. This methodology enables reliable and reproducible API quantification within human and mouse skin over time. The concentrations within lipid-rich and lipid-poor compartments, as well as total API concentration over time are quantified; these are utilized for estimates of micro- and macroscale BA and, potentially, BE.

Multi-window sparse spectral sampling stimulated Raman scattering microscopy.

Stimulated Raman scattering (SRS) is a nondestructive and rapid technique for imaging of biological and clinical specimens with label-free chemical specificity. SRS spectral imaging is typically carried out either via broadband methods, or by tuning narrowband ultrafast light sources over narrow spectral ranges thus specifically targeting vibrational frequencies. We demonstrate a multi-window sparse spectral sampling SRS (S4RS) approach where a rapidly-tunable dual-output all-fiber optical parametric oscillator is tuned into specific vibrational modes across more than 1400 cm-1 during imaging. This approach is capable of collecting SRS hyperspectral images either by scanning a full spectrum or by rapidly tuning into select target frequencies, hands-free and automatically, across the fingerprint, silent, and high wavenumber windows of the Raman spectrum. We further apply computational techniques for spectral decomposition and feature selection to identify a sparse subset of Raman frequencies capable of sample discrimination. Here we have applied this novel method to monitor spatiotemporal dynamic changes of active pharmaceutical ingredients in skin, which has particular relevance to topical drug product delivery.

Translational biophotonics with Raman imaging: clinical applications and beyond.

Clinical medicine continues to seek novel rapid non-invasive tools capable of providing greater insight into disease progression and management. Raman scattering based technologies constitute a set of tools under continuing development to address outstanding challenges spanning diagnostic medicine, surgical guidance, therapeutic monitoring, and histopathology. Here we review the mechanisms and clinical applications of Raman scattering, specifically focusing on high-speed imaging methods that can provide spatial context for translational biomedical applications.

Visualizing and quantifying antimicrobial drug distribution in tissue.

The biodistribution and pharmacokinetics of drugs are vital to the mechanistic understanding of their efficacy. Measuring antimicrobial drug efficacy has been challenging as plasma drug concentration is used as a surrogate for tissue drug concentration, yet typically does not reflect that at the intended site(s) of action. Utilizing an image-guided approach, it is feasible to accurately quantify the biodistribution and pharmacokinetics within the desired site(s) of action. We outline imaging modalities used in visualizing drug distribution with examples ranging from in vitro cellular drug uptake to clinical treatment of microbial infections. The imaging modalities of interest are: radio-labeling, magnetic resonance, mass spectrometry imaging, computed tomography, fluorescence, and Raman spectroscopy. We outline the progress, limitations, and future outlook for each methodology. Further advances in these optical approaches would benefit patients and researchers alike, as non-invasive imaging could yield more profound insights with a lower clinical burden than invasive measurement approaches used today.

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

Recent progress in plasmonic nanoparticle-based biomarker detection and cytometry for the study of central nervous system disorders.

Neurological disorders affect hundreds of millions of people around the world, are often life-threatening, untreatable, and can result in debilitating symptoms. The high prevalence of these disorders, which feature biochemical or structural abnormalities in neuronal systems, has spurned innovations in both rapid and early detection to assist in the selection of appropriate treatment strategies to improve the patients' quality of life. Plasmonic nanoparticles (PNPs), a versatile and promising class of nanomaterials, are widely utilized in numerous imaging techniques, drug delivery systems, and biomarker detection methods. Recently, PNP-based nanoprobes have attracted considerable attention for the early diagnosis of neurological disorders. Gold nanoparticles (AuNPs), with high local surface plasmon resonance (LSPR) signals, have been particularly well exploited as probes for dynamic biomarker detection, with quantification sensitivity demonstrated down to the single-molecule level. In this review, we will discuss the possibilities of PNPs in the methodological development for rapid neurological disease identification. In addition, we will also describe a new digital cytometry method that combines dark-field imaging and machine learning for precise biomarker enumeration on single cells. The aim of this review is to attract researchers working on the future development of new plasmonic nanoprobe-based strategies for the diagnosis of neurological disorders.

Characterization of Cell-Bound CA125 on Immune Cell Subtypes of Ovarian Cancer Patients Using a Novel Imaging Platform.

MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel "digital" optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK (p = 0.013), T (p < 0.001) and B cells (p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer.