We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.
Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebaceous unit of ex vivo human facial skin specimens dosed with about 2.5× daily dose using two-photon excitation fluorescence microscopy. In this study, we demonstrated another novel approach to identifying minocycline fluorescence signature using fluorescence lifetime imaging microscopy (FLIM) with phasor analysis. It was found that for a single daily dose and with FLIM, minocycline was consistently noted in the epidermis and hair follicle, with some incidence in the sebaceous gland for both 1% and 2% BPX-01. These observations corroborated with the recent success of a Phase 2b dose-finding study, with 2% BPX-01 meeting the primary endpoint of lesion reduction at week 12 with statistical significance over the vehicle.
The evaluation of complex metabolic changes of individual live cells and heterogeneous cell cultures is not feasible using traditional methods due to their destructive behavior and lack of spatial information. Two-photon excited fluorescence of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) facilitate a label-free and non-destructive evaluation of metabolic activity. This study explores the phasor approach in combination with two-photon fluorescence lifetime imaging microscopy (FLIM) as a potential method to evaluate pharmacologically induced metabolic changes that occur during the browning of adipocytes. The possibility of browning of white adipose cells is a desirable prospect for the treatment of obesity and related disorders. Here, we compared the results obtained by Fourier-based phasor analysis with the traditional exponential FLIM analysis as well as results of an extracellular flux analyzer. The alteration of glycolytic function and oxidative phosphorylation after treatment with pharmacological reagents significantly shift the contribution of each of the fluorescence lifetime components to the total fluorescence intensity. Further, we showed that the ratios of the lifetime components obtained by the phasor approach reflect the shift in mitochondrial and cytosolic NADH concentration. The phasor analysis agrees with traditional assessments, such as the optical free-to-bound NADH ratio as well as the oxygen consumption rate and extracellular acidification rate as determined by the extracellular flux analyzer. Our results support the concept that non-invasive sensing of fat metabolism and browning of fat may be possible by analyzing the fluorescence lifetime of NADH using the phasor approach.
Two-photon fluorescence lifetime imaging microscopy (FLIM) is a technique that not only probes the intensity of fluorophores, but also provides the temporal decay trace of said fluorophores on a pixel-by-pixel basis. These traces can then be transformed into the frequency domain for subsequent analysis, resulting in a scatterplot of phasor coordinates where each phasor corresponds to a single image pixel. With this in mind, it follows that individual fluorophores result in distinct clusters in the phasor plot, and a mixture of two fluorophores results in phasors that fall somewhere along a line linking the two clusters depending on the relative fluorophore concentrations. Until now, distinction of fluorescent species has relied mainly on computing the Euclidean distance between a given phasor and the mean coordinates of reference phasor clusters. However, this approach becomes inadequate in cases where one fluorophore has a much wider lifetime distribution than the other. As such, we propose the use of the Mahalanobis distance as an alternative to the Euclidean distance, as this metric additionally factors in the relative spread of each reference phasor cluster. This method has been applied to studying the oxidative response of ex vivo human skin via endogenous NADH fluorescence as it is exposed to chemical sun filters, the active ingredients in sunscreens. Given that both NADH and sun filters are fluorescent under the same excitation and emission conditions, the proposed Mahalanobis distance approach was used to distinguish the source of fluorescence in images of human skin. This allowed for the assessment of oxidative response as well as the tracking and monitoring of the sun filter formulation as it permeated throughout the skin.
One of the primary functions of human skin is to provide a mechanical barrier and interface with the outside world, owing to its unique structure and composition. Indeed, the most superficial layer of the epidermis, the stratum corneum, is a selectively porous structure composed of thin layers of protein-rich corneocytes joined together by lipids such as ceramides. The overall impermeability of the stratum corneum is crucial for homeostasis, but also hampers the penetration of beneficial topical agents into the skin. Specifically, hydrophilic compounds typically do not permeate through the epidermis, while hydrophobic compounds can readily be delivered through lipid-containing routes such as sebaceous gland ducts or the spacing between corneocytes. A proper understanding of stratum corneum structure and composition is therefore of great benefit for the design of topical formulations in order to properly optimize the delivery of active compounds to the skin. To this aim, coherent Raman scattering imaging techniques including both coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopies can be used to study the chemical composition and structure of the stratum corneum, as these modalities are sensitive to unique vibrational modes of specific chemical groups such as lipids, proteins, and water. These metrics can further be used to measure uptake and efficacy of topical compounds in order to optimize formulation design.
Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the phasor approach to FLIM. The unique signature of minocycline in FLIM phasor analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.
Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients’ skin.
While melanoma is not the most common form of skin cancer, it represents the vast majority of skin cancer-related deaths. Indeed, while combination therapies such as Dabrafenib and Trametinib have shown great promise in clinical trials for treating metastatic disease, some melanoma subtypes nevertheless develop resistances to front-line treatments. Under in vitro conditions, some metastatic human melanoma cell lines have been observed to evolve resistance to treatment while simultaneously changing color under brightfield microscopy, hinting at perturbations in pigment synthesis. The process known as melanogenesis gives rise to the two forms of melanin found in mammals: eumelanin, a dark brown/black pigment, and pheomelanin, a much more pale red/blond pigment. Interestingly, pheomelanin has been shown to contribute to the onset and development of melanoma in an ultraviolet-radiation-independent manner through a mechanism of oxidative stress. Eumelanin, on the other hand, is a known antioxidant whose chemical properties seem to shield cells against oxidative damage. To study these pigments in closer detail, nonlinear optical microscopy including coherent anti-Stokes Raman scattering (CARS) was used for the specific visualization and quantification of the relative abundance of pheomelanin and eumelanin within these treatment resistant cell lines. These microscopy toolkits provide a means to monitor changes in pigmentation in a noninvasive and non-destructive manner without the use of exogenous dyes to better understand the molecular basis of treatment resistance.
Over the past decade, nonlinear optical microscopy has seen a dramatic rise in its use in research settings due to its noninvasiveness, enhanced penetration depth, intrinsic optical sectioning, and the ability to probe chemical compounds with molecular specificity without exogenous contrast agents. Nonlinear optical techniques including two-photon excitation fluorescence (2PEF), fluorescence lifetime imaging microscopy (FLIM), second harmonic generation (SHG), coherent anti-Stokes and stimulated Raman scattering (CARS and SRS, respectively), as well as transient and sum frequency absorption (TA and SFA, respectively), have been widely used to explore the physiology and microanatomy of skin. Recently, these modalities have shed light on dermal processes that could not have otherwise been observed, including the spatiotemporal monitoring of cosmetics and pharmaceuticals. However, a challenge quickly arises when studying such chemicals in a dermatological context: many exogenous compounds have optical signatures that can interfere with the signals that would otherwise be acquired from intact skin. For example, oily solvents exhibit strong signals when probing CH2 vibrations with CARS/SRS; chemical sun filters appear bright in 2PEF microscopy; and darkly colored compounds readily absorb light across a broad spectrum, producing strong TA/SFA signals. Thus, this discussion will first focus on the molecular contrast in skin that can be probed using the aforementioned nonlinear optical techniques. This will be followed by an overview of strategies that take advantage of the exogenous compounds’ optical signatures to probe spatiotemporal dynamics while preserving endogenous information from skin.
Skin cancer, including basal cell carcinoma, squamous cell carcinoma, and melanoma, is the most common form of cancer in North America. Paradoxically, skin cancer incidence is steadily on the rise even despite the growing use of sunscreens over the past decades. One potential explanation for this discrepancy involves the sun filters in sunscreen, which are responsible for blocking harmful ultraviolet radiation. It is proposed that these agents may produce reactive oxygen species (ROS) at the site of application, thereby generating oxidative stress in skin that gives rise to genetic mutations, which may explain the rising incidence of skin cancer. To test this hypothesis, ex vivo human skin was treated with five common chemical sun filters (avobenzone, octocrylene, homosalate, octisalate, and oxybenzone) as well as two physical sun filters (zinc oxide compounds), both with and without UV irradiation. To non-invasively evaluate oxidative stress, two-photon excitation fluorescence (2PEF) and fluorescence lifetime imaging microscopy (FLIM) of the skin samples were used to monitor levels of NADH and FAD, two key cofactors in cellular redox metabolism. The relative redox state of the skin was assessed based on the fluorescence intensities and lifetimes of these endogenous cofactors. While the sun filters were indeed shown to have a protective effect from UV radiation, it was observed that they also generate oxidative stress in skin, even in the absence of UV light. These results suggest that sun filter induced ROS production requires more careful study, especially in how these reactive species impact the rise of skin cancer.
Recently, pheomelanin has been found to play a critical role in melanoma progression given its pro-oxidant chemical properties as well as its marked presence in pre-cancerous and malignant melanoma lesions, even in the absence of ultraviolet radiation. In addition, epidemiological evidence indicates a strong correlation between melanoma incidence and skin type, with the highest incidence occurring in individuals of the red-haired/fair-skinned phenotype. Interestingly, nevus count correlates well with melanoma incidence and skin type, except in the population most prone to developing melanoma, where nevus count strikingly drops. As such, a current hypothesis proposes that fair-skinned red-haired individuals, who are unable to stimulate production of eumelanin due to a mutation in MC1R in melanocytes, may actually harbor numerous “invisible”, pheomelanin-rich nevi that evade clinical detection, supporting the high incidence of melanoma in that population.
Here, we show for the very first time that melanocytes extracted from genetically modified MC1R-mutant, red-haired mice displayed bright perinuclear distributions of signal within the cells under coherent anti-Stokes Raman scattering (CARS) microscopy. Changes in pheomelanin production in siRNA knockdowns of cultured human melanoma cells were also sensed. We then successfully imaged pheomelanin distributions in both ex vivo and in vivo mouse ear skin. Finally, melanosomes within amelanotic melanoma patient tissue sections were found to show bright pheomelanin signals. This is the first time, to our knowledge, that pheomelanin has been found spatially localized in a human amelanotic melanoma sample. These pheomelanotic CARS features may be used as potential biomarkers for melanoma detection, especially for amelanotic melanomas.
We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC’s, but longer more intense pulses lead to their destruction.
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