BACKGROUND:While the detrimental effects of intense sun exposure are well-recognized, the cumulative impact of daily moderate UV exposure remains less explored. This year-long study investigated the effects of seasonal variations on facial skin, aiming at highlighting subclinical skin changes using non-invasive in vivo multiphoton microscopy. We also explored the impact of applying daily a well-balanced photoprotection formula with enhanced UVA filtration. SUMMARY:Fifty-nine European descent women (aged 55-65, Fitzpatrick phototypes II or III), participated in this year long, real-life exposure study in Paris, France. Participants were randomized into two double-blind groups: one applying a daily broad-spectrum SPF/PPD photoprotection product, the other applying the vehicle. In vivo assessments were conducted at four seasonal time points to measure the skin color and multiphoton parameters: melanin density and z-epidermal distribution, epidermal morphology, elastin and fibrillar collagen densities in the papillary dermis (first 20 µm-thick dermal sublayer below the DEJ) and their combined 3D SAAID aging index, a marker of photoaging.The vehicle group exhibited a significant increase in skin color, melanin density, elastin density and a decrease in the 3D-SAAID aging index between winter and summer, reflecting seasonal UV impact. These changes were not observed in the photoprotected group. KEY MESSAGES:This study provides in vivo evidence that real-life sun exposure, even under moderate conditions, can induce subclinical skin changes. Daily use of sunscreen with enhanced UVA protection mitigated these effects, highlighting the importance of consistent photoprotection in preventing long-term UV-induced clinical aging signs and supporting skin longevity.
BACKGROUND:The interaction between antioxidants and free radicals has become increasingly important in cosmetic research. This review is not a conventional literature overview but is primarily based on our own experimental data and methodological developments. SUMMARY:The first part describes noninvasive spectroscopic techniques, such as resonance Raman spectroscopy are highlighted as robust tools for the in vivo determination of carotenoid concentrations, serving as indirect markers of the skin's antioxidant status. The next section addresses the detection of free radicals. Electron paramagnetic resonance (EPR) spectroscopy is presented as the only direct and quantitative method for detecting free radicals in biological systems. Its application in vitro, ex vivo, and in vivo enables controlled assessment of radical formation and antioxidant efficacy, including under defined irradiation conditions. The introduction of the radical protection factor (RPF) provides a standardized parameter to compare the scavenging capacity of individual compounds and complex formulations, thereby supporting product development and claim substantiation. In the last section, in vivo and ex vivo measurements are compared. It is shown that the amounts of free radicals generated in the UV and infrared spectral ranges of solar radiation differ significantly between different skin types. For sun protection, this means that the protective effect of a sunscreen must extend across the entire spectral range of solar radiation, since 50% of the free radicals generated by the sun originate in the visible and infrared spectral ranges. KEY MESSAGES:These findings led to the development of a sunscreen important for Asian skin (skin type IV).
In recent years, multicolored tattoos have gained popularity among young individuals. However, the behavior of tattoo inks in the skin, including their distribution, accumulation, and long-term effects, remains poorly understood, partly due to limited in vivo imaging techniques. This study employs picosecond-pulsed two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) to non-invasively visualize the deposition of colored tattoo ink pigments (blue, red, green, yellow, orange, purple and black) in human skin in vivo at depths of up to ≈150 µm. The study reveals that tattoo ink agglomerates are present across various skin layers, ranging from the stratum corneum to the reticular dermis. Tattoo ink pigments were predominantly observed within dermal cells, including mast cells, macrophages, and fibroblasts, suggesting that these cells act as long-term reservoirs for the pigments. Additionally, ink residues were detected in epidermal cells, including keratinocytes, dendritic cells, and basal cells, indicating a continuous low-level release of the ink even in old tattoos. Furthermore, the analysis showed that melanin can be distinguished from certain tattoo ink pigments using phasor approach combined with TPE autofluorescence imaging. The TPE-FLIM technique enables in vivo visualization of epidermal dendritic cells and dermal fibroblasts, which are otherwise undetectable using conventional optical methods without staining. This method holds significant potential for clinical dermatology by offering insights into cellular uptake, accumulation, and long-term effects of tattoo inks, thereby supporting the management of tattoo-related complications.
BACKGROUND:A holy grail in human skin permeation is to be able to quantify how compounds and nanoparticles are absorbed, distributed, and exert their effects. Traditionally, the processes used have either been destructive or relied upon interpreting what has exited the skin after absorption, be it in in vivo plasma or urine or through ex vivo skin. Multiphoton imaging and other modalities have revolutionized this landscape. SUMMARY:In this review article, we summarize our experiences in using multiphoton tomography and fluorescence lifetime imaging in characterizing the skin absorption, distribution, and redox effects of nanoparticulate and related delivery systems. Much of our work has been focused on the absorption and safety of nano zinc oxide which started off being an apparently unsafe product to now being an FDA GRAS substance. We have found that the skin is a remarkable physical barrier to zinc oxide nanoparticles, as well as to other nanoparticles, under a wide range of formulation and method of use conditions. However, this work has only been made possible for us by the development of confocal, multiphoton, and fluorescence lifetime imaging technologies that can be safely used on human skin. KEY MESSAGES:(1) Multiphoton tomography and fluorescence lifetime imaging have allowed us, for the first time, to safely image the transport of compounds and structures in human skin in vivo and to assess the redox state of the skin after that application. (2) A key use of this instrumentation and related techniques has been to show that the sunscreen, nano zinc oxide, is generally safe when applied to human skin in various formulations and under a wide range of conditions. (3) We have also shown that characterizing nano-zinc oxide skin absorption by urinary zinc isotope excretion is misleading because, in our experience, zinc oxide nanoparticles are hydrolyzed to zinc ions which are readily absorbed into the viable epidermis and will be excreted in the urine. (4) We have also shown other nanoparticles, such as quantum dots, silver, and gold, are poorly absorbed into the skin. (5) However, our imaging does show that nanoparticles can accumulate and be difficult to wash out of skin furrows and hair follicles, creating a reservoir effect. (6) While this review has emphasized studies exploring the safety of topical ZnO-NP and related materials, we have also shown that multiphoton tomography and related techniques may be used in the quality, therapeutic, and theranostic assessment of topical products.
INTRODUCTION:Microgravity during spaceflight is increasingly associated with cutaneous impairment, yet the structural basis and underlying metabolic mechanisms remain poorly defined. Collagen, the main component of the dermal extracellular matrix, relies on de novo proline synthesis in fibroblasts. Herein, we examined whether simulated microgravity (SMG) affects collagen synthesis by altering the proline biosynthetic pathway. METHODS:SMG was established using a hindlimb unloading model in rats and a rotating flat chamber system in skin fibroblasts. Dermal morphology and collagen deposition were assessed in skin tissue, while collagen synthesis, intracellular proline levels, and pyrroline-5-carboxylate reductase 1 (PYCR1) expression were examined in fibroblasts. PYCR1 was overexpressed to evaluate its contribution to proline synthesis under SMG. RESULTS:SMG caused dermal thinning and reduced collagen deposition in rat skin, and it similarly decreased collagen production in fibroblasts. SMG also lowered intracellular proline levels in fibroblasts and downregulated the PYCR1 gene. Importantly, PYCR1 overexpression partially restored intracellular proline levels and restored collagen protein expression under SMG conditions. CONCLUSION:These findings indicate that impaired de novo proline biosynthesis, associated with PYCR1 downregulation, contributes to reduced collagen production and may underlie microgravity-induced dermal structural alterations.
Introduction: Topical corticosteroids remain the mainstay of psoriasis management but are limited by skin atrophy and barrier impairment. Developing safer, steroid-sparing therapy represents an unmet need in topical pharmacology. The glucocorticoid receptor (GR) plays a central role in epidermal homeostasis, yet selective activation by natural phytochemicals has not been systematically explored. Methods: A molecular modeling framework integrating docking, molecular dynamics simulations, and pharmacophore conservation was established to evaluate GR engagement by four phytochemicals—sakuranetin, salvianolic acid B, lithospermic acid, and magnolol. Computational findings were compared with immunohistochemical analysis of total GR and phosphorylated Ser211-GR in psoriatic epidermal specimens derived from residual archived tissues. Results: Salvianolic acid B and lithospermic acid displayed high docking affinity and stable retention of canonical GR contacts, whereas sakuranetin and magnolol exhibited moderate but consistent binding. Immunohistochemistry confirmed preserved GR abundance for all compounds, with lithospermic acid producing the greatest increase in Ser211 phosphorylation, consistent with differential receptor-associated signaling patterns. Computational and histological findings showed preliminary directional concordance between computational interaction profiles and tissue-level receptor-associated readouts. Conclusion: This study demonstrates that integrating molecular modeling with receptor-associated histological analysis may assist in prioritizing natural compounds with potential GR-engagement properties. The proposed framework provides an exploratory and reproducible strategy for receptor-engagement prioritization in topical pharmacology and may support future investigation of bioactive phytochemicals in dermatologic research.
INTRODUCTION:We investigated whether post-menopausal females receiving hormone replacement therapy (HRT) exhibit greater nitric oxide (NO)-dependent cutaneous microvascular responses than those not receiving HRT, assessed via pharmacological NO synthase (NOS) inhibition during local heating. We also examined regional differences in microvascular response between and within groups. METHODS:Twenty-one post-menopausal females (10 receiving HRT, 11 not receiving HRT) underwent assessment of cutaneous vascular conductance (CVC), expressed as a percentage of maximum vasodilation (%CVCmax), at the chest, abdomen, forearm, and calf using laser-Doppler flowmetry. Local skin temperature was raised from 33°C to 42°C for ∼30 min, followed by ∼40 min perfusion with 20 mmNG-nitro-L-arginine methyl ester (L-NAME; NOS inhibitor) using intradermal microdialysis. The NO contribution to vasodilation was defined as the difference in %CVCmax between the 42°C and L-NAME plateau. RESULTS:There were no significant differences in the NO contribution to vasodilation between groups or between sites within groups (all p > 0.107). During baseline, females not receiving HRT demonstrated 7.6 [0.8, 14.4]% higher %CVCmax at the forearm compared with abdomen (p = 0.021). During the 42°C heating plateau, this group also showed abdominal %CVCmax values that were 11.8 [0.04, 23.5]% lower than the calf (p = 0.041) and 12.7 [1.1, 24.4]% lower than the forearm (p = 0.025). Females receiving HRT demonstrated 12.0 [2.4, 21.5]% greater abdominal %CVCmax than those not receiving HRT (p = 0.014). CONCLUSION:Altogether, our findings indicate that HRT is not associated with NO-dependent cutaneous vasodilation during local heating in post-menopausal females but is associated with regional heterogeneity in cutaneous vasodilatory responses.
INTRODUCTION:The effects of ultraviolet (UV) radiation on the skin are manifold. On the one hand, it aids in the production of vitamin D and is used in phototherapy. On the other hand, it can cause cell damage leading to premature skin aging and the development of various skin pathologies, including skin cancer. Despite the importance of this issue, the development and recovery of morphological changes induced by UV irradiation are still almost unexplored. METHODS:In this in vivo study, two-photon tomography combined with fluorescence lifetime imaging was used to investigate structural changes in the skin of healthy volunteers over 30 days after a single UV irradiation (the lamp spectrum includes UVC ≈39.8%, UVB ≈7.5%, UVA ≈8.3%, and visible ≈44.4%) applied for 120 s at a total dose of 43.1 mJ/cm2. RESULTS:The results show that UV (predominantly UVC at 254 nm) irradiation leads to an increase in the nucleus/cell ratio, appearance of a bright perinuclear rim, disruption of the membrane, expanded intercellular space, and the appearance of sunburn cells in the epidermis, and a decrease in collagen type I in the dermis, which are recovered 30 days post-UV irradiation. The phasor plot approach enabled the separation of dermal cells into four clusters, allowing fluorescence lifetime analysis of resting and activated mast cells as well as M1 and M2 macrophages. We observed that UV (predominantly UVC at 254 nm) radiation can activate mast cells, leading to a decrease their fluorescence lifetime, whereas no effect was observed in macrophages. CONCLUSION:The results obtained in this in vivo study confirm that UV (predominantly UVC at 254 nm) has damaging effect on all skin layers. Two-photon tomography is an effective noninvasive method for in vivo imaging morphological changes in the skin following UV irradiation and for studying severe photodamage.
INTRODUCTION:Skin cancer is a growing global health concern, often linked to environmental carcinogens like ultraviolet radiation and chemicals. This study investigated the protective effects of sakuranetin against DMBA-induced skin cancer in mice. METHODS:A total of 24 mice were randomly divided into four groups: a control group, a disease control group, and two treatment groups that received sakuranetin orally at 10 mg/kg and 20 mg/kg post-DMBA exposure for 8 weeks. Tumorigenesis was evaluated through histopathology, followed by hematological (HB, RBCs, MCV, WBCs, PCV, and MCH), antioxidant enzyme levels (SOD, CAT, GSH), lipid peroxidation, pro-inflammatory markers (IL-1β, IL-6, TNF-α, COX-2), NF-κB, and caspase-3. Additionally, network pharmacology and molecular docking were performed. RESULTS:Treatment with sakuranetin restored the abnormal hematological parameters, restored antioxidant enzyme levels (SOD, CAT, GSH), reduced lipid peroxidation, and significantly reduced pro-inflammatory markers, NF-κB, while increased caspase-3. Histological examination confirmed reduced neoplastic changes. Sakuranetin at the dose of 20 mg/kg completely prevents tumor development. Network pharmacology identified 53 potential sakuranetin targets, and molecular docking revealed strong binding affinities with key proteins, including COX-2 (-9.2), TGF-β (-8.5), NF-κB (-8.1), caspase-3 (-6.8), and VEGF (-5.9), which are involved in cancer pathways. CONCLUSION:Sakuranetin effectively mitigated DMBA-induced skin cancer through antioxidant, anti-inflammatory, and pro-apoptotic mechanisms. These findings suggest its potential as a multi-targeted therapeutic candidate for skin cancer prevention.
Introduction: In patients with diabetes, persistent hyperglycemia causes chemical changes in serum components, increasing the incidence of various complications such as skin wounds. To better understand glucose-associated chemical stress on serum components, this study explores small-molecule nutrients to protect human dermal fibroblasts (HDFs) from glucose-rich, chemically deteriorated culture conditions. Methods: Various cell culture media with different concentrations of glucose and various test compounds were subjected to heat treatment as a chemically accelerated model of serum deterioration. The heat-treated and non-heat-treated media were compared to each other in terms of the content of glycation products and their effects on cell viability and wound healing in vitro. Results: There was no difference in the content of glycation products or their effects on cell viability between the non-heat-treated low-glucose (LG, 5.5 mm) and high-glucose (HG, 50 mm) media that were not heat treated. However, the heat-treated HG media had a higher content of glycation products and decreased cell viability compared to the heat-treated LG media. Of the 20 free amino acids, 20 amidated amino acids and various antioxidants derived from cysteine (Cys) or ascorbic acid (AA) added to the media, followed by heat treatment, only cysteinamide (C-NH2) enhanced the viability and wound healing of HDFs cultured in the heat-treated HG media. C-NH2 reduced glycation of serum proteins while forming its own glycation products, suggesting a competitive interaction with glucose-derived reactions. Conclusion: These findings provide proof-of-concept evidence that C-NH2 can protect serum components and improve fibroblast-associated wound closure under chemically stressed, glucose-rich conditions. Further studies using physiologically relevant models are required to evaluate its biological and translational significance.
INTRODUCTION:Effective management of atopic dermatitis (AD) remains challenging, as current therapies often rely on topical corticosteroids associated with adverse effects and limited long-term tolerability. METHODS:This double-blind, randomized, placebo-controlled trial investigated the effects of a 12-week oral supplementation with specific bioactive collagen peptides (BCPs, CURADERM®) on clinical symptoms and skin physiology in adults with moderate AD. Participants were randomized to receive either 5 g of BCP or placebo daily. RESULTS:BCP supplementation led to significantly lower corticosteroid rescue medication use (0.94 ± 1.42 g vs. 5.60 ± 7.82 g; p = 0.045; d = 0.847) and an earlier reduction in pruritus, as shown by improvements in the 5-D Pruritus Scale at week 4 (p = 0.024, d = 1.49). Favourable effects were also observed for key physiological parameters of the skin, including maintenance of physiological pH (4.5-5.5) and improved hydration of lesional areas (p = 0.040, d = 1.44). No treatment-related adverse events occurred. CONCLUSION:Taken together, these findings provide first placebo-controlled evidence that oral BCP supplementation supports skin barrier recovery, stabilizes physiological skin function, and accelerates symptom relief in mild-to-moderate AD. Confirmation of these promising results in larger trials is warranted to validate treatment effects.
INTRODUCTION:Sensitive skin is a common skin condition that impairs quality of life and is characterised by unpleasant sensations to normally non-irritating stimuli. Restoration of skin barrier integrity can relieve symptoms, and accumulating evidence indicates a contributory role of the skin microbiome not only in barrier function but also as a potential modulator in sensitive skin. Ceramide-containing emollients improve barrier function and may modulate microbial communities via restoration of stratum corneum lipids, hydration, and immune regulation. METHODS:In this randomized, double-blind, vehicle-controlled trial, an emollient containing Ceramide NP C15 was evaluated for its effects on sensitive skin symptoms and skin barrier function. Fifty participants applied the study products in a split-body design for six weeks. Primary assessments included skin physiology and symptom burden. Furthermore, skin microbiota changes were explored using flow cytometry (FC)-based bacterial profiling and 16s rRNA gene sequencing. RESULTS:Both the Ceramide NP C15 formulation and the vehicle were associated with improvements in subjective symptoms. In participants with impaired skin barrier, treatment with the ceramide formulation was associated with a significant reduction in transepidermal water loss. FC- and 16S-based analyses indicated modest, treatment-associated changes in skin microbiota composition including an early and sustained increase in microbial evenness and a significant enrichment of Bifidobacterium. CONCLUSION:In this exploratory, randomized controlled study, a Ceramide NP C15-containing emollient was associated with improvements in skin barrier function. Exploratory microbiome analyses suggested treatment-associated changes in microbial community characteristics, which should prompt further investigations in their clinical relevance for sensitive skin.
INTRODUCTION:Silymarin (SM), an extract from the fruits of the milk thistle (Silybum marianum L., Asteraceae), has pleiotropic effects on human health. SM's biological activity assigns to its constitutive polyphenols, mainly flavonolignans. SM has become increasingly interesting for dermal applications. There are currently several commercial dermatological preparations available on the market containing SM or its dominant flavonolignan silybin (SB) as the active ingredients. The metabolic transformation of compounds in skin including SM's polyphenols has only been marginally investigated. Therefore, this study focused on the metabolization of SM and its pure polyphenols (SB, isosilybin [ISB], 2,3-dehydrosilybin [DHSB], silychristin [SC], silydianin [SD], and taxifolin [TA]) in the skin. METHODS:Human skin ex vivo (HSE) and normal human dermal fibroblasts (NHDF) were used as experimental models. For cell treatment, a concentration of 10 μmol L-1 (pure compounds) or 4.825 mg L-1 (SM) was used. For HSE treatment, 50 μmol L-1 (pure compounds) or 24.124 mg L-1 (SM) was used. RESULTS:The penetration of studied polyphenols into the skin correlated with their lipophilicity (DHSB>ISB>SB>SC>SD>TA). The bioavailability of SM's polyphenols was relatively high, and thus the substances can be expected to modulate biological processes in the region close to their application site. SM's polyphenol metabolites of the first and second phase of biotransformation were identified in NHDF and HSE. CONCLUSION:In HSE, the metabolic transformation of SM's components and its pure polyphenols was more extensive, as skin fragments are a more complex cellular and metabolically active system. Human skin ex vivo seems to be a useful tool for studying compounds biotransformation at topical application.
INTRODUCTION:Skin hydration, which is defined by the hydration index and the spatial distribution of water, represents one of the key parameters reflecting the functional state of the skin. Although various optical methods have been proposed to study the hydration process, these techniques are often limited by a shallow probing depth, insufficient depth resolution, or low spatial and temporal resolution, which precludes a comprehensive assessment of the skin response to topical pharmaceutical agents. METHODS:We present an approach for the quantitative assessment and visualization of skin dehydration and rehydration processes with high depth and temporal resolutions. The proposed method is based on reconstructing the scattering coefficient from the optical coherence tomography data. We used this approach to quantify the effective skin dehydration depth and rehydration time in rats ex vivo following the topical application of 70% ethanol and an ethanolic solution of the betamethasone dipropionate, glucocorticosteroid, in both the presence and absence of ultrasound exposure (1 MHz, 0.5 W/cm2, 2 min). RESULTS:Ethanol was shown to induce reversible dehydration of the upper skin layers (168 ± 88 µm) with the recovery occurring within 21 ± 3 min. Ultrasound treatment of the ethanol application area resulted in the enhancement of its penetration depth up to 262 ± 63 µm and reduced the rehydration time to 14 ± 3 min. The addition of betamethasone dipropionate to the ethanol solution further increased the dehydration depth and delayed rehydration process. Visualization of the obtained data as heatmaps enabled a quantitative evaluation of the spatiotemporal changes in the skin. CONCLUSION:The proposed approach proved to be effective for investigating water diffusion processes in the skin and would facilitate the analysis of dynamic changes in both healthy and pathological tissues, as well as the assessment of the effects of various topical chemical and physical treatments. This work holds significant implications for both basic and practical researches at the interface between optics and biomedicine.
INTRODUCTION:Recent progress in supramolecule research has led to a surge in its application in cosmetic products. However, comprehensive investigation of the potential benefits, especially differentiating biological activities and associating that with physical-chemical properties, remains to be established. The current study aimed to investigate the supramolecular salicylic acid-betaine (Supra-SA-B) compared to its physical mixture (SA+B) for effects on epidermal homeostasis and topical insult potential. METHODS:The structure of supra-SA-B and SA+B was characterized by a powder X-ray diffractometer. Skin tolerance was evaluated by applying SA, Supra-SA-B, and SA+B to human keratinocytes or SkinEthic™ reconstructed human epidermis (RHE) models. Inflammatory cytokines were assessed by Luminex assay. Histological morphology was evaluated by hematoxylin-eosin staining, with aquaporin-3 (AQP3) and Ki67 measured by immunofluorescence. RNA sequencing characterized overall biological response. RESULTS:Characteristic peaks remained the same for Supra-SA-B after freeze-drying verified that their crystalline structures were retained in the current solution. Supra-SA-B treated groups demonstrated significantly higher viabilities compared to SA or SA+B. With 18-h of treatment, Supra-SA-B treated group released fewer cytokines. In addition, Supra-SA-B demonstrated a better hydration effect with increased AQP3 expression and more organized epidermal structure. Consistent result was observed in the RNA transcriptomic profiles of RHE models. Supra-SA-B did not induce major transcriptome drift as compared to SA or SA+B, with less evident negative impact on loricrin, and late cornified envelope gene expression. Hyaluronic acid synthase boosting was maintained. CONCLUSION:Supra-SA-B ameliorated skin tolerance risk for SA, enhanced hydration with less insult on barrier functions, suggesting promising potential for cosmetic application.
BACKGROUND:Sequential application of topical dermatologic medications - defined as applying two or more agents to the same anatomical site within a short interval - is common in dermatologic therapy. Despite widespread clinical use, the pharmacologic consequences of application order, timing, and vehicle interactions on percutaneous absorption remain incompletely characterized. SUMMARY:This review synthesizes clinical, in vivo human volunteer, and mechanistic in vitro and ex vivo evidence evaluating the impact of sequential topical application on cutaneous drug absorption and bioavailability. A qualitative literature search identified twelve eligible studies, including randomized controlled trials, in vivo human studies, and experimental diffusion and visualization models. Across heterogeneous study designs, sequential application was not pharmacologically neutral. Alterations in percutaneous absorption were driven by changes in stratum corneum barrier properties and vehicle or excipient interactions rather than formulation class alone. Sequencing effects were most pronounced when the first-applied product induced keratolysis, hydration or occlusion, or lipid film formation, or when vehicle redissolution and redistribution altered the effective treated surface area. In barrier-compromised conditions, such as atopic dermatitis, changes in application order were less likely to translate into measurable clinical effects. Fixed-combination formulations generally demonstrated more consistent and predictable cutaneous delivery than free sequential regimens. KEY MESSAGES:Sequential topical application can significantly influence percutaneous absorption through barrier modification and vehicle interactions. Pharmacokinetic effects are context-dependent and cannot be inferred solely from formulation type. Mechanism-informed sequencing and judicious selection of compatible vehicles may improve predictability of topical drug delivery, highlighting the need for standardized, pharmacologically focused studies to guide application order and timing.
INTRODUCTION:This study investigates the transdermal permeation efficiency and traceability of fluorescent nanodiamond-conjugated small extracellular vesicles (FND-sEVs) derived from umbilical cord mesenchymal stem cells. METHODS:First, the sEVs were isolated and characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis, confirming their typical cup-shaped morphology and size distribution (62.5-141.1 nm) consistent with canonical sEV properties. Subsequently, FNDs were conjugated to sEVs, forming FND-sEVs, whose composite structure - featuring a high-electron-density FND core surrounded by a vesicular sEV membrane - was verified by TEM. Conjugation efficiency was then validated via confocal microscopy, showing complete colocalization between FND fluorescence and Alexa Fluor 488-labeled wheat germ agglutinin-tagged sEVs. Transdermal assessment was conducted using a skin tissue model, with magnetically modulated fluorescence (MMF) spectroscopy applied for background-free quantification. RESULTS:Leveraging the unique nitrogen-vacancy centers in FNDs (fluorescence lifetime ∼20 ns), MMF eliminated interference from skin autofluorescence (∼3 ns) and enabled direct quantification in tissue digests without pre-separation. FND-sEVs were discovered to remain predominantly in the supernatant, with a measurable fraction permeating skin tissue layers, while their presence in the filtrate was negligible, thereby indicating limited transdermal passage. CONCLUSION:Collectively, these findings establish FND-sEVs as a traceable platform for transdermal studies and highlight MMF as a robust tool for quantifying nanoscale vesicle distributions in complex biological matrices.
INTRODUCTION:Optical clearing (OC) of biological objects is a promising technique with broad potential in clinical and diagnostic applications, as it enhances imaging depth, spatial resolution, and contrast of acquired images or spectra. The aim of this study was to study the effects of the common optical clearing agents (OCAs) glycerol, fructose, dimethyl sulfoxide (DMSO), and iohexol (Omnipaque®) solutions at various concentrations and treatment durations on the stratum corneum (SC) of intact skin using confocal Raman microspectroscopy (CRM). METHODS:The pig ear was selected for study. Various OCAs were applied topically on the skin, including water solutions of DMSO (100%), fructose (50%), Omnipaque® (90% and 100%), glycerol (40%, 60%, 80%, 90%, and 100%). Ex vivo measurements were carried out on porcine ear skin using a Model 3510 SCA confocal Raman microscope (RiverD International B.V., Rotterdam, The Netherlands). RESULTS:It was found that a 25%-25%-50% water-ethanol-fructose solution permeated the SC at all tested exposure times (5, 15, 30, and 45 min). Similarly, a mixture of 80% glycerol and 20% DMSO, applied for 45 min, also demonstrated effective penetration through the SC. Enhanced OC was achieved by combining OCAs with DMSO, ethanol, or distilled water. The highest OC efficiency was observed with a 25%-25%-50% water-ethanol-fructose solution and a mixture of 90% Omnipaque® with 10% DMSO, both applied for 5 min, resulting in a 4.1- and 4.2-fold increase, respectively, in signal intensity at a skin depth of 80 µm. It was shown that the optical properties of the skin can be controlled at a depth of approx. 80 μm. Almost all used OCAs, which are not occlusive from the conventional viewpoint, have an occlusion effect on the SC. CONCLUSION:The findings of this study contribute to the identification of optimal OCA combinations - namely, a 25%-25%-50% water-ethanol-fructose solution and a mixture of 90% Omnipaque® with 10% DMSO, each applied for 5 min - for potential in vivo applications. Moreover, the results provide a strong foundation for future multimodal investigations aimed at developing advanced methods for visualizing biological tissues and organs and analyzing their biophysical parameters, in conjunction with controlled OC.
INTRODUCTION:Heart failure (HF) is an age-related condition that complicates heart disease. Currently, the number of patients with HF continues to increase, representing a significant burden on the healthcare system. Prognostication of patients with HF may be performed with modern spectroscopic approaches. METHODS:This study proposes to utilize conventional spontaneous Raman spectroscopy and autofluorescence for the in vivo analysis of skin tissues to create a prognosis for patients with HF. We collected skin spectral data from 160 HF patients. Twenty-nine patients died during the 1-year observation period. After the preprocessing of spectral data, we proposed a classification model for the prediction of mortality. This model utilizes projection on latent structures combined with discriminant analysis. Stability of the model was demonstrated during division of the data into training and test. RESULTS:Analysis of full spectral data provided only 55% accuracy in a 1-year mortality prediction, while analysis of autofluorescence and Raman spectral data provides 66% and 70% accuracy, respectively. The combination of autofluorescence and Raman spectroscopy provides an accuracy of 74% (68% sensitivity and 80% specificity) and an receiver operating characteristic-area under the curve of 0.80 for the prediction of 1-year mortality in patients with HF. The most important Raman bands for the prediction of 1-year mortality appeared at 1,086-1,180, 1,320, 1,465, and 1,770 cm-1. CONCLUSION:Raman spectroscopy could be a powerful tool for the prognosis of patients with HF; however, further studies on a larger cohort are required to demonstrate the applicability of the proposed spectral in vivo analysis.
INTRODUCTION:Knowing thicknesses of skin-tissue layers within the depths of several hundred micrometers from the surface is highly important for numerous biomedical applications - cosmetology, treatment of wounds and burns, characterization of various lesions, etc. In this regard, optical coherence tomography (OCT) with its ability to noninvasively enable visualization depth of the order of 1 mm with a resolution of the order of several micrometers offers exceptional diagnostic possibilities largely unavailable to other techniques. This explains high interest to OCT utilization in dermatology. METHODS:This study demonstrates that appropriate physics-based processing of OCT data allows one to objectively reveal and automatically estimate thicknesses of morphological skin layers, even if they are not visible as optical layers with differing intensity in initial structural OCT scans. To this end, we apply recently developed efficient methods of spatially resolved estimation of the scattering coefficient µs to locally characterize the scattering strength of scatterers in the tissue. Another useful parameter termed speckle contrast (SCI) characterizes fluctuations of scattering strengths of scatterers and their clustering. Mapping parameters µs and SCI enable differentiation of morphological skin layers, although in structural OCT scans they cannot be clearly delineated even by experts. RESULTS:Analysis of parameters µs and SCI for OCT data in vivo acquired in various localizations for women and man of various ages confirmed the possibility to clearly segment three medically significant skin layers: (i) stratum corneum, (ii) living-cell layer of epidermis, and (iii) upper dermis layer even if in the initial OCT scans these layers are hardly discernible. The present study is intentionally limited to healthy facial skin, for which significant variations in the layer thicknesses were demonstrated for various age groups, as well as in quite close localizations for the same person. CONCLUSION:These findings underscore high potential of in vivo OCT imaging supplemented with the analysis of speckle features and optical attenuation parameter for objective real-time differentiation of morphological skin layers, which is critically important for numerous applications (surgical reconstruction of skin wounds and/or burn injuries, controlled drug delivery in targeted skin layers, choice of therapy). Similar approach may be extended for diagnosing various skin diseases/pathologies.
