Highly Robust Multilamellar Lipid Vesicles Generated through Intervesicular Self-Assembly Mediated by Hydrolyzed Collagen Peptides.

Despite the well-known advantages of lipid vesicles for drug and gene delivery, structural instability limits their practical applications and requires strictly regulated conditions for transport and storage. Chemical crosslinking and in situ polymerization have been suggested to increase the membrane rigidity and dispersion stability of lipid vesicles. However, such chemically modified lipids sacrifice the dynamic nature of lipid vesicles and obfuscate their in vivo metabolic fates. Here, we present highly robust multilamellar lipid vesicles through the self-assembly of preformed, cationic large unilamellar vesicles (LUVs) with hydrolyzed collagen peptides (HCPs). The cationic LUVs undergo vesicle-to-vesicle attachment and structural reorganization through polyionic complexation with HCPs, resulting in the formation of multilamellar collagen-lipid vesicles (MCLVs). The resulting MCLVs exhibit excellent structural stability against variations in pH and ionic strength and the addition of surfactants. Particularly, the MCLVs maintain their structural stability against repeated freeze-thaw stresses, proving the unprecedented stabilization effect of biological macromolecules on lipid lamellar structures. This work provides a practically attractive technique for the simple and quick fabrication of structurally robust lipid nanovesicles without covalent crosslinkers, organic solvents, and specialized instruments.

Chronic infrared-A irradiation-induced photoaging of human dermal fibroblasts from different donors at physiological temperature.

BACKGROUND: In this study, we examined cellular responses to acute and chronic IRA irradiation at mild and natural levels of exposure in two types of human fibroblasts, each isolated from a different donor, at physiological temperature (34°C). METHOD: Two types of human dermal fibroblasts (derived from a 20- and 50-year-old women, respectively) were exposed to different repeat numbers of IRA exposure (3, 6, 10, and 14 times; 42 mW/cm2 ) at a frequency of 3-4 times per week (4 h per irradiation). Cellular responses to acute and chronic IRA irradiation were examined by reactive oxygen species (ROS) level, apoptotic signals, cellular morphology, and collagen level. RESULTS: We demonstrated that chronic IRA irradiation-induced severe cellular damage, including prolonged cell proliferation, increased intracellular ROS levels, activated cellular apoptosis, and elongated cell morphology, whereas acute IRA irradiation had negligible effects at 34°C. In addition, it was evident that the degree of cellular damage due to IRA irradiation differed according to the type of fibroblasts. CONCLUSIONS: Considering the severe cellular damage induced by chronic IRA irradiation without heat, continuous exposure of skin to IRA irradiation during daily life may be harmful enough to induce photoaging.

Electric fields regulate cellular elasticity through intracellular Ca2+ concentrations.

Cellular elasticity is a key factor related to a broad range of physiological and pathological processes. The elasticity of a single cell has thus emerged as a potential biomarker to characterize the cellular state. Both internal and external stimuli affect cellular elasticity, and changes in elasticity can cause alterations in cellular characteristics or function. The application of electric fields (EFs) is a promising method that can be used to change cellular elasticity; however, the mechanisms underlying its effect remain unknown. Here, we demonstrate EFs-induced elasticity changes in human dermal fibroblasts and discuss the underlying mechanism related to actin polymerization. Cellular elasticity increases after EF (50 mV/mm) stimulation, reaching a maximum at 30 min before decreasing between 30 and 120 min. The cellular elasticity under EF stimulation, regardless of stimulation time, is higher than that of the control. F-actin regulates the elasticity of cells through gelsolin activation. We show changes in intracellular Ca2+ caused by EFs, which induced gelsolin activation and F-actin content changes. This result demonstrates a series of processes in which external electrical stimulation conditions regulate cellular elasticity.

In vivo determination of the Infrared-A protection factor on human skin.

BACKGROUND: Chronic exposure to infrared A (IR-A) irradiation causes photoaging. However, daily or acute exposure to IR-A rarely induces erythema or pigmentation. Thus, evaluation of the physiological changes taking place on the skin surface is insufficient for clinical investigations. MATERIALS AND METHODS: We fabricated a novel device to obtain the IR-A protection factor (IPF) on human skin. This device consists of an artificial light source that mimics the actual IR-A intensity of sunlight, and a spectrophotometer to measure the spectral reflectance on the skin surface. The IPF can be determined by measuring the difference in spectral reflectance on the skin before and after the use of products and can be verified by the statistical criterion. A validation study was performed using different light intensities and two experimenters. Finally, we monitored the IPF on 12 commercial cosmetics. RESULTS: After considering the IPF and L*-values, we selected the optimal sample and performed a validation study. Neither the intensity of IR-A irradiation or the experimenters significantly affected the IPF. 12 commercial products exhibited their own IPF values and were verified by statistical criteria, with one exception. CONCLUSION: The present IPF evaluation method was concluded to be robust and reliable. This method is simple and safe for the subjects, and could be helpful for the development of IR-A protection products and the confirmation of product performances.

Electric field-induced changes in biomechanical properties in human dermal fibroblasts and a human skin equivalent.

PURPOSE: An electric field (EF) can be used to change the mechanical properties of cells and skin tissues. We demonstrate EF-induced elasticity changes in human dermal fibroblasts (HDFs) and a human skin equivalent and identify the underlying principles related to the changes. METHODS: HDFs and human skin equivalent were stimulated with electric fields of 1.0 V/cm. Change in cellular elasticity was determined by using atomic force microscopy. Effects of EF on the biomechanical and chemical properties of a human skin equivalent were analyzed. In cells and tissues, the effects of EF on biomarkers of cellular elasticity were investigated at the gene and protein levels. RESULTS: In HDFs, the cellular elasticity was increased and the expression of biomarkers of cellular elasticity was regulated by the EF. Expression of the collagen protein in the human skin equivalent was changed by EF stimulation; however, changes in density and microstructure of the collagen fibrils were not significant. The viscoelasticity of the human skin equivalent increased in response to EF stimulation, but molecular changes were not observed in collagen. CONCLUSIONS: Elasticity of cells and human skin equivalent can be regulated by electrical stimulation. Especially, the change in cellular elasticity was dependent on cell age.

A novel in vivo test method for evaluating the infrared radiation protection provided by sunscreen products.

BACKGROUND: Infrared radiation (IR) exposure generates reactive oxygen species and induces matrix metalloproteinase-1 expression in human skin. Moreover, while not as acute as ultraviolet radiation, repeated infrared irradiation can result in the photoaging of skin. Broad-spectrum sunscreens can protect skin from IR, but no human in vivo test methods for the evaluation of sunscreens' IR protection effect have been developed. We aimed to develop such a method. MATERIALS AND METHODS: We included 155 Korean subjects in our three-part clinical study. The IR reflectance of subjects' skin was measured using a benchtop model of an IR light source and a reflectance measuring probe. We measured the IR reflectance in relation to skin color and hydration level to set up our experimental conditions. We then calculated the infrared protection factors (IPFs) of cosmetic emulsions as the IR reflectance ratio between cosmetic sunscreen-applied skin and non-sunscreen-applied skin and assessed the relationship between IPFs and the amount of sunscreen ingredients. Finally, this method was validated using several commercial sunscreen cosmetics. RESULTS: Skin color and hydration level did not influence the IR reflectance of subjects' skin. The IPFs of cosmetic sunscreens showed a positive correlation with the amount of inorganic sunscreen ingredients. CONCLUSION: In this study, we developed a simple, fast, and ethically acceptable human in vivo test method for evaluating the IPFs of cosmetic sunscreens.

Ultra-trace level determination of diquat and paraquat residues in surface and drinking water using ion-pair liquid chromatography with tandem mass spectrometry: a comparison of direct injection and solid-phase extraction methods.

Direct injection and solid-phase extraction methods for the determination of diquat and paraquat in surface and drinking water were developed using liquid chromatography with tandem mass spectrometry. The signal intensities of analytes based on six ion-pairing reagents were compared with each other, and 12.5 mM nonafluoropentanoic acid was selected as the best suited amongst them. A clean-up method was developed using Oasis hydrophilic-lipophilic balance; this was compared to the direct injection method, with respect to limits of detection, interference, precision, and accuracy. Limits of quantification of diquat and paraquat were 0.03 and 0.01 µg/L using the direct injection method, and 0.002 and 0.001 µg/L using the hydrophilic-lipophilic balance method. When the hydrophilic-lipophilic balance method was used to analyze target compounds in 114 surface water and 30 drinking water samples, paraquat and diquat were detected within a concentration range of 0.001-0.12 and 0.002-0.038 µg/L in surface water, respectively. When the direct injection method was used to analyze target compounds in the same samples, the detected concentrations of paraquat and diquat were within 25% in samples being >0.015 µg/L using the hydrophilic-lipophilic balance method. The liquid chromatography with tandem mass spectrometry method using direct injection can thus be used for routine monitoring of paraquat and diquat in surface and drinking water.

Liposome-assisted penetration and antiaging effects of collagen in a 3D skin model.

BACKGROUND: Collagen is a major component of the extracellular matrix that supports the epidermal layers of the skin; thus, many strategies have been made to enhance the topical delivery of collagen for antiaging purposes. In addition, our previous study indicated that liposome can help the penetration of active ingredients into the skin. AIMS: To produce stable collagen-encapsulated liposomes to improve the topical delivery of collagen. METHODS: Collagen-encapsulated liposomes were fabricated using high-pressure homogenization method. The colloidal stability and adhesion ability were confirmed using dynamic light scattering, and spectrofluorophotometer, respectively. Keratinocyte differentiations of 3D skin before and after treatment with collagen-encapsulated liposomes were confirmed by real-time PCR. RESULTS: In comparison with native collagen, collagen-encapsulated liposomes enhanced collagen retention in artificial membranes by twofold, even after repeated washings with water. In addition, real-time PCR results indicated that 3D skin treated with collagen-encapsulated liposomes exhibited higher levels of collagen, keratin, and involucrin, even after ethanol treatment. CONCLUSION: Liposomes could serve as efficient delivery vehicles for collagen, thereby enhancing its antiaging effects.

Multilayered collagen-lipid hybrid nanovesicles for retinol stabilization and efficient skin delivery.

Lipid-based nanocarriers have been extensively utilized for the solubilization and cutaneous delivery of water-insoluble active ingredients in skincare formulations. However, their practical application is often limited by structural instability, leading to premature release and degradation of actives. Here we present highly robust multilamellar nanovesicles, prepared by the polyionic self-assembly of unilamellar vesicles with hydrolyzed collagen peptides, to stabilize all-trans-retinol and enhance its cutaneous delivery. Our results reveal that the reinforced multilayer structure substantially enhances dispersion stability under extremely harsh conditions, like freeze-thaw cycles, and stabilizes the encapsulated retinol. Interestingly, these multilamellar vesicles exhibit significantly lower cytotoxicity to human dermal fibroblasts than their unilamellar counterparts, likely due to their smaller particle number per weight, minimizing potential disruptions to cellular membranes. In artificial skin models, retinol-loaded multilamellar vesicles effectively upregulate collagen-related gene expression while suppressing the synthesis of metalloproteinases. These findings suggest that the robust multilamellar vesicles can serve as effective nanocarriers for the efficient delivery and stabilization of bioactive compounds in cutaneous applications.

Enhanced stability, formulations, and rheological properties of nanoemulsions produced with microfludization for eco-friendly process.

HYPOTHESIS: Eco-friendly processes that are emerging around the world require mass production of low-energy, low-cost nanoemulsions. The process involving the high-concentrated nanoemulsions and diluting them with a large amount of solvent can certainly save the cost; however, not much detailed research has been conducted on the stability mechanism and rheological characteristics of high-concentrated nanoemulsions. EXPERIMENTS: In this study, we produced nanoemulsions via the microfluidization (MF) process, comparing their dispersion stability and rheological characteristics with macroemulsions across various oil and surfactant concentrations. Droplet mobility and dispersion stability depended on these concentrations, with Asakura-Osawa-type attractive depletion considering interparticle interaction's role in stability changes. We investigated nanoemulsions' long-term stability based on turbidity and droplet size changes over four weeks, proposing a stability diagram showing four different states depending on emulsification conditions. FINDINGS: We explored the microstructure of emulsions under varying mixing conditions, observing their effects on droplet mobility and rheological properties. We monitored changes in rheology, turbidity, and droplet size over 4 weeks, establishing stability diagrams for macro- and nanoemulsions. The stability diagrams revealed that the stability of emulsions are sensitively dependent on the droplet size, concentrations, surfactant cocentrations and the strcture of coexistent phases in case of macroscopic segregation are significantly different depending on the droplet sizes. We identified their respective stability mechanisms and discovered the relationship between stability and rheological properties for highly concentrated nanoemulsion.

Microencapsulation of alcohol solvents and high-content actives for efficient transdermal delivery.

The barrier function of the skin in effectively protecting the underlying tissue from the surrounding environment makes it challenging to achieve the efficient transdermal delivery of actives. Herein, we report on alcohol-solvent-encapsulated microcapsules to achieve enhanced skin efficacy. We show that using palm oil as the shell material allows for the microencapsulation of a broad range of alcohol solvents, including ethanol and dipropylene glycol (DPG), as well as on-demand release. Moreover, clinical trials reveal that the high-content actives in microcapsules result in enhanced skin efficacy, and the presence of DPG effectively mediates the transdermal delivery of these actives without causing any skin irritation. We envision that the alcohol-solvent microencapsulation strategy outlined in this work offers new possibilities in cosmetics, food, and drug delivery systems.

Hydrangea serrata Hot Water Extract and Its Major Ingredient Hydrangenol Improve Skin Moisturization and Wrinkle Conditions via AP-1 and Akt/PI3K Pathway Upregulation.

Hydrangea serrata is a plant grown in Korea and Japan with a particular natural compound, hydrangenol. H. serrata has been researched for its anti-fungal properties, and ability to attenuate allergies and promote muscle growth. Its ability to reduce skin dryness is poorly understood. For that reason, we investigated whether H. serrata hot water extracts (Hs-WE) can moisturize keratinocytes. In clinical studies (Approval Code: GIRB-21929-NY and approval Date: 5 October 2021), skin wrinkles and skin moisturizing levels were improved in subjects applying 0.5% Hs-WE compared to the placebo group. We confirmed the components of Hs-WE from the LC/MS-MS analysis. Hs-WE and hydrangenol did not show cytotoxicity in HaCaT cells at all concentrations. Cell growth was also promoted by Hs-WE (5-20 µg/mL) and hydrangenol (15-60 µM) in a wound healing assay. Skin moisturizing factors were upregulated by the presence of Hs-WE or hydrangenol, and the hyaluronidases (HYAL) were inhibited at the mRNA level. Meanwhile, COL1A1 was increased by the presence of Hs-WE or hydrangenol. MAPK, AP-1, and Akt/PI3k signaling proteins, which are associated with cell proliferation and moisturizing factors, were increased by the administration of Hs-WE and hydrangenol. Has-1, 2, and 3 levels were enhanced via JNK when using the inhibitors of MAPK proteins and Hs-WE and hydrangenol, respectively. Taken together, Hs-WE could be used as cosmeceutical materials for improving skin conditions.

Multilamellar ceramide core-structured microvehicles with substantial skin barrier function recovery.

This study introduces a multilamellar ceramide core-structured microvehicle platform for substantial skin barrier function recovery. Our approach essentially focused on fabricating bacterial cellulose nanofiber (BCNF)-enveloped ceramide-rich lipid microparticles (CerMPs) by solidifying BCNF-armored oil-in-water Pickering emulsions. The oil drops consisted of Ceramide NP (a phytosphingosine backbone N-acylated with a saturated stearic acid) and fatty alcohols (FAs) with a designated stoichiometry. The thin BCNF shell layer completely blocked the growth of ceramide molecular crystals from the CerMPs for a long time. The CerMP cores displayed a multilamellar structure wherein the interlayer distance and lateral packing could be manipulated using FAs with different alkyl chain lengths. The CerMPs remarkably lowered the trans-epidermal water loss while restoring the structural integrity of the epidermis in damaged skin. The results obtained herein highlight that the CerMP system provides a practical methodology for developing various types of skin-friendly formulations that can strengthen the skin barrier function.

Skin Penetration Enhancer-Incorporated Lipid Nanovesicles (SPE-LNV) for Skin Brightening and Wrinkle Treatment.

In this work, we utilize skin penetration enhancers (SPEs) such as ceramide and fatty acids in lipid nanovesicles to promote the transdermal delivery of active ingredients. These SPE-incorporated lipid nanovesicles (SPE-LNV) interact with the constituents of skin's outermost stratum corneum (SC) layer, enabling even niacinamide and adenosine with high water solubility to effectively permeate through, leading to enhanced skin efficacy. We demonstrate by both in vitro and in vivo skin permeation studies that the SPE-LNV formulation containing both ceramide and fatty acids (LNV-CF) exhibits deeper penetration depth and faster permeation rate compared to conventional lipid nanovesicles (LNV) without SPE as well as LNV-C with only ceramide. Moreover, in vivo clinical trials were also performed to confirm that LNV-CF most effectively mediates the delivery of niacinamide and adenosine, resulting in a substantial decrease in melanin index as well as skin wrinkle compared to the control groups. We envision that the strategy of incorporating both ceramide and fatty acids in lipid nanovesicles offers a simple and convenient route for the rapid and effective delivery of water-soluble active ingredients across the skin barrier layer.

Clinical evaluation of the brightening effect of chitosan-based cationic liposomes.

BACKGROUND: Cationic liposomes can enhance the permeability of drugs in 3-D skin. Chitosan is considered a safe material for percutaneous delivery; thus, this study uses chitosan-incorporated cationic liposomes. AIMS: This study investigated the improvement in skin brightness, melanin, and melasma after treatment niacinamide-incorporated chitosan cationic liposomes. METHODS: A skin brightening agent, niacinamide, was formulated into cationic liposomes to facilitate percutaneous absorption and was clinically tested in 21 Korean female subjects. Cationic liposomes were prepared using a high-pressure homogenizer after mixing an oil phase containing lecithin and cholesterol and an aqueous phase containing niacinamide and chitosan. RESULTS: The cationic liposomes exhibited stability over 28 days, with a particle size of 255-275 nm and zeta potential of 10-14 mV. Cationic liposomes containing niacinamide and a control formulation were applied to the left and right side of the face, respectively, twice daily for 28 days. Skin brightness, melanin index, and area of melasma were significantly enhanced where cationic liposomes were used, in comparison with formulations without cationic liposomes, demonstrating a 1.38-2.08-fold improvement. CONCLUSION: Thus, we established that chitosan liposomes augmented the percutaneous absorption of niacinamide and improved the appearance of the skin.

Oleanolic Acid Protects the Skin from Particulate Matter-Induced Aging.

The role of particulate matter (PM) in health problems including cardiovascular diseases (CVD) and pneumonia is becoming increasingly clear. Polycyclic aromatic hydrocarbons, major components of PM, bind to aryl hydrocarbon receptor (AhRs) and promote the expression of CYP1A1 through the AhR pathway in keratinocytes. Activation of AhRs in skin cells is associated with cell differentiation in keratinocytes and inflammation, resulting in dermatological lesions. Oleanolic acid, a natural component of L. lucidum, also has anti-inflammation, anticancer, and antioxidant characteristics. Previously, we found that PM10 induced the AhR signaling pathway and autophagy process in keratinocytes. Here, we investigated the effects of oleanolic acid on PM10-induced skin aging. We observed that oleanolic acid inhibits PM10-induced CYP1A1 and decreases the increase of tumor necrosis factor- alpha and interleukin 6 induced by PM10. A supernatant derived from keratinocytes cotreated with oleanolic acid and PM10 inhibited the release of matrix metalloproteinase 1 in dermal fibroblasts. Also, the AhR-mediated autophagy disruption was recovered by oleanolic acid. Thus, oleanolic acid may be a potential treatment for addressing PM10-induced skin aging.

Biocompatible Wax-Based Microcapsules with Hermetic Sealing for Thermally Triggered Release of Actives.

We present a microfluidic approach that utilizes temperature-responsive and biocompatible palm oil as the shell material in microcapsules to simultaneously achieve hermetic sealing as well as on-demand temperature-triggered release of the encapsulated actives. Unlike common paraffin waxes (e.g., eicosane), microcapsule shells comprising palm oil do not form pores or cracks during freezing and provide a hermetic seal, a nearly perfect seal that separates the core containing the actives from the surrounding environment over a prolonged period of time. This allows effective isolation and protection of complex cargoes such as small molecules with high diffusivity, strong acids, and cosmetic actives including niacinamide. Moreover, the palm oil shell melts above the defined melting temperature, allowing the on-demand release of the encapsulated actives. Furthermore, palm oil is biocompatible, is edible, and leaves a minimal footprint when used in personal care and cosmetic products, offering new perspectives in the design of microcapsules for cosmetic applications.

Ultraviolet light screen using cholesteric liquid crystal capsules on the basis of selective reflection.

Sunscreen can protect human skin from sunlight by decreasing exposure to ultraviolet (UV) light, specifically UV-B and UV-A. In this study, a new type of UV screen system is proposed using cholesteric liquid crystal (CLC) capable of selectively reflecting UV-A within the human skin temperature range of 32-36 °C. Polycaprolactone (PCL) capsules with CLC mixture which had a helical chiral pitch corresponding to the wavelength of UV light were made by a solvent evaporation method. The average diameter of the capsules was about 34 µm. Consequently, it was confirmed that the CLC mixture (COC : CN = 80 : 20) could reflect UV-A light over 350-380 nm within the human skin temperature range. Also, it was confirmed that the CLC/PCL microcapsules could block UV light over 290-400 nm by about 6%.

Comparison Study of the Effects of Cationic Liposomes on Delivery across 3D Skin Tissue and Whitening Effects in Pigmented 3D Skin.

Charged phospholipids are employed to formulate liposomes with different surface charges to enhance the permeation of active ingredients through epidermal layers. Although 3D skin tissue is widely employed as an alternative to permeation studies using animal skin, only a small number of studies have compared the difference between these skin models. Liposomal delivery strategies are investigated herein, through 3D skin tissue based on their surface charges. Cationic, anionic, and neutral liposomes are formulated and their size, zeta-potential, and morphology are characterized using dynamic light scattering and cryogenic-transmission electron microscopy (cryo-TEM). A Franz diffusion cell is employed to determine the delivery efficiency of various liposomes, where all liposomes do not exhibit any recognizable difference of permeation through the synthetic membrane. When the fluorescence liposomes are applied to 3D skin, considerable fluorescence intensity is observed at the stratum cornea and epithelium layers. Compared to other liposomes, cationic liposomes exhibit the highest fluorescence intensity, suggesting the enhanced permeation of liposomes through the 3D skin layers. Finally, the ability of niacinamide (NA)-incorporated liposomes to suppress melanin transfer in pigmented 3D skin is examined, where cationic liposomes exhibit the highest degree of whitening effects.

Highly Sustainable and Completely Amorphous Hierarchical Ceramide Microcapsules for Potential Epidermal Barrier.

As a main component of the stratum corneum, ceramides can construct protective lamellae to provide an epidermal barrier against dehydration or external microorganisms. However, as ceramide molecules can easily form the isolated crystalline phase through self-assembly due to the amphipathic nature of bioactive lipids, the effective incorporation of ceramides into liquid media is the remaining issue for controlled release. Here, we report an unprecedented effective strategy to fabricate a completely amorphous and highly sustainable hierarchical ceramide polymer microcapsule for promising epidermal barrier by using the interpenetrating and cooperative self-construction of conical amphiphiles with a different critical packing parameter. The self-constructed amorphous architecture of ceramides in polymer microcapsule is achieved by the facile doping of conical amphiphiles and subsequent in situ polymerization of shell polymer in the core-shell geometry. It is experimentally revealed that an irregular cooperative packing structure formed by adaptive hydrophobic-hydrophilic interactions of cylindrical ceramides and conical amphiphiles in the confined microcapsule geometry enables a completely amorphous morphology of ceramides to be realized during the spontaneous encapsulation process. Furthermore, this elegant approach affords a highly dispersible and uniform hierarchical amorphous ceramide microcapsule with a greatly enhanced long-term stability compared to conventional crystalline ceramides.